Antibodies against g-csfr and uses thereof

ABSTRACT

The present disclosure provides proteins comprising antigen binding domains of antibodies that bind to human granulocyte-colony stimulating factor receptor.

RELATED APPLICATION

The present application claims priority from U.S. Provisional PatentApplication No. 61/496,351 entitled “Antibodies against G-CSFR and usesthereof” filed on 13 Jun. 2011, the entire contents of which are herebyincorporated by referenced.

SEQUENCE LISTING

The present application is filed together with a Sequence Listing inelectronic form. The entire contents of the Sequence Listing are herebyincorporated by reference.

FIELD

The present disclosure relates to antibodies that bind togranulocyte-colony stimulating factor receptor (G-CSFR) receptor anduses thereof, e.g., in therapy.

BACKGROUND

Granulocyte colony-stimulating factor (G-CSF) is a major regulator ofgranulocyte production. G-CSF is produced by bone marrow stromal cells,endothelial cells, macrophages, and fibroblasts, and production isinduced by inflammatory stimuli. G-CSF acts through the G-CSF receptor(G-CSFR), which is expressed on early myeloid progenitors, matureneutrophils, monocytes/macrophages, T and B lymphocytes and endothelialcells. Mice deficient in G-CSF or the G-CSFR exhibit marked neutropenia,demonstrating the importance of G-CSF in steady-state granulopoiesis.However, G-CSF appears to be dispensable for emergency granulopoiesis,e.g., in response to an infection. G-CSF increases the production andrelease of neutrophils, mobilizes hematopoietic stem and progenitorcell, and modulates the differentiation, lifespan, and effectorfunctions of mature neutrophils. G-CSF may also exert effects onmacrophages, including expansion of monocyte/macrophage numbers,enhancement of phagocytic function, and regulation of inflammatorycytokine and chemokine production. G-CSF has also been shown to mobilizeendothelial progenitor cells and induce or promote angiogenesis.

While G-CSF is used therapeutically, e.g., to treat neutropenia and/ormobilize hematopoietic stem cells, it also has negative actions in someconditions, e.g., inflammatory conditions and/or cancer. For example,administration of G-CSF exacerbates rheumatoid arthritis (RA), murinecollagen-induced arthritis (CIA) and a passive transfer model of CIA inrats. G-CSF has been found in the serum and synovial fluid of RApatients. Furthermore, interleukin (IL)-1 and tumor necrosis factor α(TNFα), which are found at increased levels in patients suffering fromRA, induce the production of G-CSF by human synovial fibroblasts andchondrocytes. Mice deficient in G-CSF are resistant to the induction ofacute and chronic inflammatory arthritis.

G-CSF has also been shown to play a role in multiple sclerosis (MS). Forexample, G-CSF enhances adhesion of an auto-reactive T cell line modelof MS to extracellular matrix as effectively as interferon γ and TNFα,which are known to exacerbate MS symptoms. Moreover, G-CSF deficientmice are resistant to development of experimental autoimmuneencephalomyelitis (EAE).

G-CSF and G-CSFR have also been tied to cancer, with studies showingthat this signaling pathway contributes to chemotherapy resistance,growth, survival, invasiveness and metastasis of various cancers.Moreover, G-CSF has been shown to induce to angiogenesis, a processimportant in the development of solid tumors.

It will be clear to the skilled person from the foregoing, that there isa need in the art for reagents that reduce the signaling of G-CSFthrough the G-CSFR. Exemplary agents will be suitable for use astherapeutics, e.g., to treat or prevent a G-CSF-mediated condition.

SUMMARY

The present inventors have produced a class of proteins comprisingantibody binding sites (e.g., Fabs and antibodies) that bind to humanG-CSFR (hG-CSFR) and potently neutralize G-CSF signaling, e.g., preventformation of granulocytes from CD34⁺ bone marrow cells and/or preventcell proliferation in response to G-CSF and/or reduce or preventneutrophilia induced by administration of G-CSF. A class of proteinsidentified by the inventors also cross-react cynomolgus monkey G-CSFR(cynoG-CSFR), which facilitates pre-clinical studies with the proteins.A class of proteins identified by the inventors bind to hG-CSFR withhigh affinity. A class of proteins identified by the inventors are humanantibodies, which are suitable for treatment of a variety of conditions.

The present disclosure provides a protein comprising an antigen bindingsite of an antibody, wherein the antigen binding site binds to hG-CSFRand neutralizes G-CSF signaling, and wherein the protein inhibits growthof colony forming units—granulocytes (CFU-G) from CD34⁺ bone marrowcells grown in the presence of G-CSF with an IC₅₀ of at least about 0.2nM. For example, the IC₅₀ is 0.1 nM or less, for example, 0.09 nM orless, or 0.08 nM or less, or 0.07 nM or less, or 0.06 nM or less or 0.05nM or less. In one example, the IC₅₀ is 0.04 nM or less. In anotherexample, the IC₅₀ is 0.02 nM or less. Methods for assessing IC₅₀ of aprotein in such an assay are described herein. For example, the IC₅₀ isdetermined in the presence of 10 ng/ml of hG-CSF.

In one example, the IC₅₀ is determined by culturing CD34⁺ bone marrowcells in the presence of 10 ng/ml stem cell factor and 10 ng/ml hG-CSF.For example, the cells are grown in semi-solid cell culture medium. Inone example, the CFU-G are enumerated after 14 days of culture.

The present disclosure additionally or alternatively provides a proteincomprising an antigen binding site of an antibody, wherein the antigenbinding site binds to both human and cynomolgus monkey G-CSFR with asimilar affinity and neutralizes G-CSF signaling. Such proteins areadvantageous since they facilitate pre-clinical studies in non-humanmammals.

In one example, the affinity of the protein is determined using abiosensor, e.g., by surface plasmon resonance. For example, the ligandbinding region or soluble hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc orcyno-G-CSFR-Fc is immobilized and the affinity of the protein of thedisclosure is determined.

The present disclosure additionally provides a provides a proteincomprising an antigen binding site of an antibody, wherein the antigenbinding site binds specifically to the same epitope in hG-CSFR as thatbound by C1.2 (comprising a heavy chain variable region (V_(H))comprising a sequence set forth in SEQ ID NO: 2 and a light chainvariable region (V_(L)) comprising a sequence set forth in SEQ ID NO: 3)or C1.2G (comprising a V_(H) comprising a sequence set forth in SEQ IDNO: 4 and a V_(L) comprising a sequence set forth in SEQ ID NO: 5).

The present disclosure additionally or alternatively provides a proteincomprising an antigen binding site of an antibody, wherein (i) theprotein competitively inhibits binding of C1.2 (comprising a V_(H)comprising a sequence set forth in SEQ ID NO: 2 and a V_(L) comprising asequence set forth in SEQ ID NO: 3) or C1.2G (comprising a V_(H)comprising a sequence set forth in SEQ ID NO: 4 and a V_(L) comprising asequence set forth in SEQ ID NO: 5) to hG-CSFR; (ii) the proteinneutralizes G-CSF signaling; and (iii) the level of binding of theprotein to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for any one of:

(a) the arginine at position 287 of SEQ ID NO:1;

(b) the histidine at position 237 of SEQ ID NO:1;

(c) the methionine at position 198 of SEQ ID NO:1;

(d) the tyrosine at position 172 of SEQ ID NO:1;

(e) the leucine at position 171 of SEQ ID NO:1; or

(f) the leucine at position 111 of SEQ ID NO:1

is lower than the level of binding of the protein to a polypeptide ofSEQ ID NO: 1.

The present disclosure additionally or alternatively provides a proteincomprising an antigen binding site of an antibody, wherein (i) theprotein competitively inhibits binding of C1.2 (comprising a V_(H)comprising a sequence set forth in SEQ ID NO: 2 and a V_(L) comprising asequence set forth in SEQ ID NO: 3) or C1.2G (comprising a V_(H)comprising a sequence set forth in SEQ ID NO: 4 and a V_(L) comprising asequence set forth in SEQ ID NO: 5) to hG-CSFR; (ii) the proteinneutralizes G-CSF signaling; and (iii) preferentially binds to apolypeptide of SEQ ID NO: 1 relative to its ability to bind to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for anyone of:

(a) the arginine at position 287 of SEQ ID NO:1;

(b) the histidine at position 237 of SEQ ID NO:1;

(c) the methionine at position 198 of SEQ ID NO:1;

(d) the tyrosine at position 172 of SEQ ID NO:1;

(e) the leucine at position 171 of SEQ ID NO:1; or

(f) the leucine at position 111 of SEQ ID NO:1.

The present disclosure additionally or alternatively provides a proteincomprising an antigen binding site of an antibody, wherein (i) theprotein binds to hG-CSFR; (ii) the protein neutralizes G-CSF signaling;and (iii) the level of binding of the protein to a polypeptide of SEQ IDNO: 1 in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO:1

(b) the histidine at position 237 of SEQ ID NO:1;

(c) the methionine at position 198 of SEQ ID NO:1;

(d) the tyrosine at position 172 of SEQ ID NO:1;

(e) the leucine at position 171 of SEQ ID NO:1; or

(f) the leucine at position 111 of SEQ ID NO:1

is lower than the level of binding of the protein to a polypeptide ofSEQ ID NO: 1.

The present disclosure additionally or alternatively provides a proteincomprising an antigen binding site of an antibody, wherein (i) theprotein binds to hG-CSFR; (ii) the protein neutralizes G-CSF signaling;and (iii) preferentially binds to a polypeptide of SEQ ID NO: 1 relativeto its ability to bind to a polypeptide of SEQ ID NO: 1 in which analanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO:1

(b) the histidine at position 237 of SEQ ID NO:1;

(c) the methionine at position 198 of SEQ ID NO:1;

(d) the tyrosine at position 172 of SEQ ID NO:1;

(e) the leucine at position 171 of SEQ ID NO:1; or

(f) the leucine at position 111 of SEQ ID NO:1.

In one example, the level of binding of the protein to the polypeptidecomprising the alanine substitution is reduced by at least about 10 foldor 20 fold or 50 fold or 100 fold or 150 fold or 200 fold compared tothe binding of the protein to the polypeptide of SEQ ID NO: 1.Preferably, the level of binding of the protein to the polypeptidecomprising the alanine substitution is reduced by at least about 50fold. Preferably, the level of binding of the protein to the polypeptidecomprising the alanine substitution is reduced by at least about 60fold.

In one example, the antigen binding site of the protein does notdetectably bind to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for the arginine at position 287 of SEQ ID NO: 1.

In one example, the level of binding is assessed using a biosensor,e.g., by surface plasmon resonance. For example, the protein isimmobilized and the level of binding to a polypeptide of SEQ ID NO: 1 orto a form of the polypeptide comprising an alanine substitution isdetermined

Additional forms of a polypeptide comprising the amino acids of SEQ IDNO: 1 with or without other substitutions bound or not significantlybound or not detectably bound by a protein of the present disclosure aredescribed herein and are to be taken to apply mutatis mutandis to thepresent examples of the disclosure.

In one example, the antigen binding site cross-reacts with:

(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted forthe lysine at position 167 of SEQ ID NO: 1; and/or(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substitutedfor the histidine at position 168 of SEQ ID NO: 1.

In one example, the antigen binding site additionally cross-reacts witha polypeptide of SEQ ID NO: 1 in which an alanine is substituted for theleucine at position 169 of SEQ ID NO: 1

In one example, the protein competitively inhibits the binding of C1.2(comprising a V_(H) comprising a sequence set forth in SEQ ID NO: 2 anda V_(L) comprising a sequence set forth in SEQ ID NO: 3) or C1.2G(comprising a V_(H) comprising a sequence set forth in SEQ ID NO: 4 anda V_(L) comprising a sequence set forth in SEQ ID NO: 5) to one or moreof:

(i) a polypeptide of SEQ ID NO: 1 in which an alanine is substituted forthe lysine at a position 167 of SEQ ID NO: 1; and/or(ii) a polypeptide of SEQ ID NO: 1 in which an alanine is substitutedfor the histidine at position 168 of SEQ ID NO: 1.

In one example, a protein described herein according to any examplebinds to an epitope comprising residues within one or two or three orfour regions selected from 111-115, 170-176, 218-234 and/or 286-300 ofSEQ ID NO: 1.

In one example, upon binding of a protein described herein according toany example to a polypeptide of SEQ ID NO: 1 and cleavage usingprotelolytic enzymes remains bound to one or two or three or fourpeptides comprising or consisting of amino acids 111-115 of SEQ ID NO: 1or amino acids 170-176 of SEQ ID NO: 1 or amino acids 218-234 of SEQ IDNO: 1 or amino acids 286-300 of SEQ ID NO: 1.

In one example, the protein binds to a conformational epitope.

The present disclosure additionally or alternatively provides a proteinthat binds to hG-CSFR and neutralizes G-CSF signaling, the proteincomprising at least one of:

(i) a V_(H) comprising a complementarity determining region (CDR) 1comprising a sequence set forth in SEQ ID NO: 6, a CDR2 comprising asequence set forth in SEQ ID NO: 7 and a CDR3 comprising a sequence atleast about 55% identity to the sequence set forth in SEQ ID NO: 8;(ii) a V_(H) comprising a sequence at least about 80%, such as 85% or90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%identical to a sequence set forth in SEQ ID NO: 2 and/or 4;(iii) a V_(L) comprising a CDR1 comprising a sequence set forth in SEQID NO: 9, a CDR2 comprising a sequence set forth in SEQ ID NO: 10 and aCDR3 comprising a sequence at least about 33% identity to the sequenceset forth in SEQ ID NO: 11; and(iv) a V_(L) comprising a sequence at least about 80%, such as 85% or90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%identical to a sequence set forth in SEQ ID NO: 3 and/or 5.

Such a protein can comprise any one or more of the functional activitiesdescribed herein, e.g., preferential binding to a polypeptide of SEQ IDNO: 1 relative to the level of binding of a polypeptide of SEQ ID NO: 1in which an alanine is substituted for any one of:

(a) the arginine at position 287 of SEQ ID NO:1

(b) the histidine at position 237 of SEQ ID NO:1;

(c) the methionine at position 198 of SEQ ID NO:1;

(d) the tyrosine at position 172 of SEQ ID NO:1;

(e) the leucine at position 171 of SEQ ID NO:1; or

(f) the leucine at position 111 of SEQ ID NO:1.

In one example, the percentage identity at (ii) is at least about 95%.

In one example, the percentage identity at (iv) is at least about 94%.

In one example, differences between the recited sequence and the proteinare substitutions.

The skilled artisan will be capable of determining sites for mutationsto a protein of the disclosure, e.g., within a framework region of avariable region containing protein. Moreover, the inventors haveidentified numerous sites in a V_(H) CDR3 and a V_(L) CDR3 that can bemutated as well as numerous mutations that maintain activity of aprotein of the disclosure. For example a mutation, e.g., a substitutionis within one or more (e.g., 2 or 3 or 4) of the four C-terminalresidues of HCDR3 and/or one or more (e.g., 2 or 3 or 4 or 5 or 6) ofthe N-terminal or C-terminal residues of LCDR3. In one example, theN-terminal five amino acids of V_(H) CDR3 are LGELG. In one example, thethree N-terminal amino acids of V_(L) CDR3 are QQS and/or the threeC-terminal amino acids of V_(L) CDR3 are PLT.

In one example, the V_(H) comprises a CDR3 comprising a sequenceLGELGX₁X₂X₃X₄, wherein:

X₁ is selected from the group consisting of tryptophan, glutamine,methionine, serine, phenylalanine, glutamic acid and histidine and/or isa neutral amino acid, such as tryptophan, glutamine or methionine, forexample, the amino acid is tryptophan;X₂ is an amino acid selected from the group consisting of phenylalanine,tyrosine, methionine, serine, glycine and isoleucine, for example isphenylalanine, tyrosine, methionine or serine, for example, the aminoacid is phenylalanine;X₃ is an amino acid selected from the group consisting of aspartic acid,methionine, glutamine, serine, leucine, valine, arginine and histidine,for example, is proline, glutamic acid, alanine, leucine, phenylalanineor tyrosine, for example, the amino acid is aspartic acid; andX₄ is any amino acid or an amino acid selected from the group consistingof proline, glutamic acid, alanine, leucine, phenylalanine, tyrosine,threonine, asparagine, aspartic acid, serine, glycine, arginine, andlysine, for example, the amino acid is proline.

In one example, the V_(L) comprises a CDR3 comprising a sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉, wherein:

X₁ is an amino acid selected from the group consisting of glutamine,glutamic acid, histidine, alanine and serine and/or is a hydrophilicamino acid, such as glutamine or glutamic acid, for example, the aminoacid is glutamine;X₂ is an amino acid selected from the group consisting of glutamine,valine, phenylalanine, asparagine and glutamic acid, for example, theamino acid is glutamine;X₃ is an amino acid selected from the group consisting of serine andglycine, for example, the amino acid is serine;X₄ is an amino acid selected from the group consisting of tryptophan,methionine, phenylalanine, tyrosine, isoleucine and leucine, forexample, the amino acid is tryptophan or tyrosine;X₅ is any amino acid or an amino acid selected from the group consistingof glutamic acid, methionine, glutamine, tryptophan, serine, valine,asparagine, glycine, alanine, arganine, histidine, tyrosine, lysine andthreonine, for example, the amino acid is serine;X₆ is an amino acid selected from the group consisting of tyrosine,methionine, isoleucine and threonine, for example, the amino acid ismethionine, tyrosine or threonine;X₇ is an amino acid selected from the group consisting of proline,alanine, histidine, glycine and lysine, for example the amino acid isproline;X₈ is an amino acid selected from the group consisting of leucine,glutamine, methionine, alanine, phenylalanine, isoleucine, lysine,histidine and glycine, for example, the amino acid is leucine;X₉ is any amino acid or an amino acid selected from the group consistingof threonine, phenylalanine, tyrosine, methionine, lysine, serine,histidine, proline, tryptophan, isoleucine, glutamine, glycine andvaline, for example, the amino acid is threonine.

The present disclosure additionally or alternatively provides a protein(e.g., an antibody) that binds to hG-CSFR and neutralizes G-CSFsignaling, the protein comprising at least one variable region of anantibody selected from the group consisting of:

(i) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 2;(ii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:3;(iii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:4;(iv) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:5;(v) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:14;(vi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:15;(vii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:16;(viii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:17;(ix) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:18;(x) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:19;(xi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:20;(xii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:21;(xiii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:22;(xiv) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:23;(xv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:24;(xvi) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:25;(xvii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:26;(xviii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 27;(xix) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:28;(xx) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:29;(xxi) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:30;(xxii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:31;(xxiii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 32;(xxiv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:33;(xxv) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:34;(xxvi) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:35;(xxvii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 36;(xxviii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 37;(xxix) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:38;(xxx) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:39;(xxxi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:40;(xxxii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 41;(xxxiii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 42;(xxxiv) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 43;(xxxv) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:44;(xxxvi) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 45;(xxxvii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 46;(xxxviii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 47;(xxix) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:48;(xl) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:49;(xli) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:50;(xlii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:51;(xliii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 52;(xliv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:53;(xlv) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:54;(xlvi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:55;(xlvii) a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 56;(xlviii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 57;(xlix) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:58;(l) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:59;(li) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:60;(lii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:61;(liii) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:62; and(liv) a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:63.

In one example, a protein described herein comprises at least a V_(H)and a V_(L), wherein the V_(H) and V_(L) bind to form a Fv comprising anantigen binding domain. The skilled artisan will understand that theantigen binding domain comprises the binding site of the antibody.

In one example, the V_(H) and the V_(L) are in a single polypeptidechain. For example, the protein is:

-   (i) a single chain Fv fragment (scFv);-   (ii) a dimeric scFv (di-scFv); or-   (iii) at least one of (i) and/or (ii) linked to a constant region of    an antibody, Fc or a heavy chain constant domain (C_(H)) 2 and/or    C_(H)3.

In one example, the V_(L) and V_(H) are in separate polypeptide chains.

For example, the protein is:

-   (i) a diabody;-   (ii) a triabody;-   (iii) a tetrabody;-   (iv) a Fab;-   (v) a F(ab′)₂;-   (vi) a Fv; or-   (vii) one of (i) to (vi) linked to a constant region of an antibody,    Fc or a heavy chain constant domain (C_(H)) 2 and/or C_(H)3.

The foregoing proteins (described in the previous two lists) can also bereferred to as antigen binding domains of antibodies.

In one example, the protein is an antibody. In one example, the antibodyis a naked antibody.

In one example, a protein is chimeric, de-immunized, humanized, human orprimatized.

In one example, the protein or antibody is human.

(i) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 2and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO: 3;(ii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO: 5;(iii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:15 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:14;(iv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:16;(v) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:17;(vi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:18;(vii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:20 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:19;(viii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:4 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:21;(ix) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:22;(x) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 24and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:23;(ix) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:25;(x) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:26;(xi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:28 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:27;(xii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:4 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:29;(xiii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:31 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:30;(xiv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:33 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:32;(xv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:34;(xvi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:36 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:35;(xvii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:38 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:37;(xviii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 40 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 39;(xix) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:42 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:41;(xx) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:43;(xxi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:45 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:44;(xxii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:4 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:46;(xxiii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 4 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 47;(xxiv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:51 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:48;(xxv) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:51 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:50;(xxvi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:53 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:52;(xxvii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 4 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 54;(xxviii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 55 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 5;(xxix) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:57 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:56;(xxx) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:59 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:58;(xxxi) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO:61 and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO:60;(xxxii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 4 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 62;(xxxiii) a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 4 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 63; and(xxxix) a V_(H) comprising three CDRs of a V_(H) set forth in any one ormore of (i) to (xxxiii) and a V_(L) comprising three CDRs of a V_(L) setforth in any one or more of (i) to (xxxiii).

Sequences of exemplary V_(H) and V_(L) are described in Table 3, whereinthe recited V_(H) or V_(L) CDR3 sequence is substituted for thecorresponding sequence in the V_(H) or V_(L) of C1.2 or C1.2G asdescribed herein.

In one example, the present disclosure provides an antibody that bindsto hG-CSFR and neutralizes G-CSF signaling, the antibody comprising:

(i) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 2and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO: 3;or(ii) a V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 4and a V_(L) comprising an amino acid sequence set forth in SEQ ID NO: 5.

The present disclosure additionally or alternatively provides anantibody comprising a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 64 and a light chain comprising an amino acidsequence set forth in SEQ ID NO: 65. In one example, the antibody bindsto hG-CSFR and neutralizes G-CSF signaling.

The present disclosure additionally or alternatively provides anantibody comprising a heavy chain comprising an amino acid sequence setforth in SEQ ID NO: 68 and a light chain comprising an amino acidsequence set forth in SEQ ID NO: 65. In one example, the antibody bindsto hG-CSFR and neutralizes G-CSF signaling.

The present disclosure additionally or alternatively provides anantibody comprising one heavy chain comprising an amino acid sequenceset forth in SEQ ID NO: 64 and one heavy chain comprising an amino acidsequence set forth in SEQ ID NO: 68 and two light chains comprising anamino acid sequence set forth in SEQ ID NO: 65. In one example, theantibody binds to hG-CSFR and neutralizes G-CSF signaling.

Reference herein to a protein or antibody that “binds to” hG-CSFRprovides literal support for a protein or antibody that “bindsspecifically to” hG-CSFR.

In one example, a protein or antibody described herein does notsignificantly bind to mouse G-CSFR and/or does not detectably bind tomouse G-CSFR.

In one example, a protein or antibody described herein according to anyexample competitively inhibits binding of C1.2 and/or C1.2G to hG-CSFRor a cell expressing same or SEQ ID NO: 1 or a soluble hG-CSFR (e.g.,comprising amino acids 1-311 of SEQ ID NO: 1 fused to a Fc region of anantibody).

In one example, a protein or antibody described herein binds to a ligandbinding region of hG-CSFR and a ligand binding region of cynoG-CSFR withsimilar affinity. In one example, the protein binds to soluble hG-CSFRand soluble cynoG-CSFR with similar affinity. In one example, theprotein binds to a polypeptide comprising SEQ ID NO: 1 and to apolypeptide comprising SEQ ID NO: 67 with similar affinity. In oneexample, the protein binds to hG-CSFR-Fc and cynoG-CSFR-Fc as describedherein with similar affinity. In one example, the affinity is at leastabout 2 nM, for example, at least about 1.5 nM, such as at least about1.2 nM, 1.1 nM or 1 nM. In one example, the 0.5 nM, such as, at leastabout 0.46 nM or 0.45 nM or 0.40 nM or 0.39 nM. In another example, theaffinity is at least about 0.1 nM, such as at least about 0.09 nM, forexample, at least about 0.08 nM. In one example, the level of binding isassessed using a biosensor, e.g., by surface plasmon resonance. Forexample, the ligand binding region or soluble hG-CSFR or solublecynoG-CSFR or hG-CSFR-Fc or cyno-G-CSFR-Fc is immobilized and the levelof binding to a protein of the disclosure is determined

In another example, the protein of the disclosure is immobilized on, forexample, a biosensor and the level of binding of the ligand bindingregion or soluble hG-CSFR or soluble cynoG-CSFR or hG-CSFR-Fc orcyno-G-CSFR-Fc is determined For example, the level of binding to theextracellular domain of hG-CSFR or cynoG-CSFR is determined. Inaccordance with this example, the affinity of the protein for theextracellular domain of cynoG-CSFR is at least about 1 nM, such as atleast about 0.9 nM, for example, at least about 0.75 nM. For example,the affinity is at least about 0.7 nM, such as at least about 0.6 nM,for example, at least about 0.5 nM. In one example, the affinity isabout 0.5 nM. Alternatively, or additionally, the affinity of theprotein for the extracellular domain of hG-CSFR is at least about 7 nMor 6 nM or 5 nM, such as at least about 4 nM, for example, at leastabout 3 nM, e.g., at least about 2.5 nM. For example, the affinity is atleast about 2.4 or 2.5 nM.

The present disclosure also provides antigen binding domains or antigenbinding fragments of the foregoing antibodies.

In one example, a protein or antibody as described herein comprises aconstant region of an IgG4 antibody or a stabilized constant region ofan IgG4 antibody. In one example, the protein or antibody comprises anIgG4 constant region with a proline at position 241 (according to thenumbering system of Kabat (Kabat et al., Sequences of Proteins ofImmunological Interest Washington D.C. United States Department ofHealth and Human Services, 1987 and/or 1991)).

The C-terminal lysine of the heavy chain constant region of a wholeantibody of the disclosure may be removed, for example, duringproduction or purification of the antibody, or by recombinantlyengineering the nucleic acid encoding a heavy chain of the antibody.Accordingly, whole antibodies may comprise antibody populations with allC-terminal lysine residues removed, antibody populations with noC-terminal lysine residues removed, and antibody populations having amixture of antibodies with and without the C-terminal lysine residue. Insome examples, the antibody populations may additionally compriseantibodies in which the C-terminal lysine residue is removed in one ofthe heavy chain constant regions. Similarly, a composition of wholeantibodies may comprise the same or a similar mix of antibodypopulations with or without the C-terminal lysine residue.

In one example, the stabilized constant region comprises a sequence fromposition 119 to position 445 of SEQ ID NO: 64. In one example, thestabilized constant region comprises a sequence from position 119 toposition 444 of SEQ ID NO: 68. In one example a protein or antibody asdescribed herein or a composition of a protein or antibody as describedherein, comprises a heavy chain constant region, including a stabilizedheavy chain constant region, comprising a mixture of sequences fully orpartially with or without the C-terminal lysine residue.

In one example, an antibody of the disclosure comprises a V_(H)disclosed herein linked or fused to an IgG4 constant region orstabilized IgG4 constant region (e.g., as discussed above) and the V_(L)is linked to or fused to a kappa light chain constant region.

The present disclosure also provides a protein or antibody whichinhibits G-CSF-induced proliferation of a BaF3 cell expressing hG-CSFRwith an IC₅₀ of at least about 6 nM. For example, the IC₅₀ is 5.9 nM orless. In another example, the IC₅₀ is 2 nM or less or 1 nM or less or0.7 nM or less or 0.6 nM or less or 0.5 nM or less. In one example, theIC₅₀ is determined by culturing BaF3 cells (e.g. about 2×10⁴ cells) inthe presence of about 0.5 ng/ml hG-CSF, e.g., for about 48 hours. In oneexample, the proliferation of the BaF3 cells is determined by measuring3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)reduction.

The present disclosure also provides a protein or antibody whichinhibits G-CSF-induced proliferation of a BaF3 cell expressing hG-CSFRwith an IC₅₀ of at least about 10 μg/ml. For example, the IC₅₀ is 5μg/ml or less. In another example, the IC₅₀ is 3 μg/ml or less or 2μg/ml or less or 1 μg/ml or less. In one example, the IC₅₀ is about 0.8μg/ml. In one example, the IC₅₀ is determined by culturing BaF3 cells(e.g. about 1×10⁴ cells) in the presence of about 10 ng/ml hG-CSF, e.g.,for about 48 hours. In one example, the proliferation of the BaF3 cellsis determined by measuring ³H-thymidine incorporation.

In one example, a protein or antibody of the disclosure binds to asoluble hG-CSFR comprising amino acid 1-311 of SEQ ID NO: 1 expressed asa fusion with an antibody Fc region (hG-CSFR-Fc) with an affinity of atleast about 1.5 nM. For example, the affinity is at least about 0.5 nMor 0.4 nM or 0.35 nM or 0.33 nM. In one example, the affinity of theprotein is determined using a biosensor, e.g., by surface plasmonresonance. For example, the hG-CSFR-Fc is immobilized and the affinityof the protein of the disclosure is determined.

In one example, a protein or antibody of the disclosure binds to hG-CSFRexpressed on the surface of a cell at an affinity of at least about 1nM, for example, at least about 0.5 nM, such as, at least 0.4 nM, forexample, at least 0.3 nM, such as, at least 0.2 nM.

In one example, a protein as described herein according to any exampleis capable of reducing the number of neutrophils in circulation when orif administered to a cynomolgus monkey. For example, the protein reducesthe number of neutrophils in circulation when or if administered to acynomolgus monkey at a dose of between 0.05 m/kg-30 mg/kg, preferablybetween 0.1 mg/kg-10 mg/kg, e.g., administered at a dose of 0.1 mg/kg or1 mg/kg or 2 mg/kg or 5 mg/kg or 10 mg/kg. For example, the proteinreduces the number of neutrophils in circulation when or if administeredfollowing administration of G-CSF or filgrastim or a PEGylated formthereof, e.g., when or if the protein is administered about 12 hoursafter administration of G-CSF or filgrastim or a PEGylated form thereof.In one example, the reduction is a 2 fold or 3 fold reduction. In oneexample, the neutrophils are reduced about 10-24 hours, e.g., about 12hours following administration.

In one example, a protein or antibody as described herein is isolatedand/or recombinant.

In one example, a protein or antibody of the disclosure is conjugated toanother compound, for example, a detectable label or a compound thatextends the half-life of the protein or antibody, such as polyethyleneglycol or an albumin binding protein.

The present disclosure also provides a nucleic acid encoding the proteinor antibody of the present disclosure.

In one example, such a nucleic acid is included in an expressionconstruct in which the nucleic acid is operably linked to a promoter.Such an expression construct can be in a vector, e.g., a plasmid.

In examples of the disclosure directed to single polypeptide chainproteins, the expression construct may comprise a promoter linked to anucleic acid encoding that polypeptide chain.

In examples directed to multiple polypeptide chains that form a protein,an expression construct comprises a nucleic acid encoding a polypeptidecomprising, e.g., a V_(H) operably linked to a promoter and a nucleicacid encoding a polypeptide comprising, e.g., a V_(L) operably linked toa promoter.

In another example, the expression construct is a bicistronic expressionconstruct, e.g., comprising the following operably linked components in5′ to 3′ order:

(i) a promoter(ii) a nucleic acid encoding a first polypeptide;(iii) an internal ribosome entry site; and(iv) a nucleic acid encoding a second polypeptide,wherein the first polypeptide comprises a V_(H) and the secondpolypeptide comprises a V_(L), or vice versa.

The present disclosure also contemplates separate expression constructsone of which encodes a first polypeptide comprising a V_(H) and anotherof which encodes a second polypeptide comprising a V_(L). For example,the present disclosure also provides a composition comprising:

(i) a first expression construct comprising a nucleic acid encoding apolypeptide comprising a V_(H) operably linked to a promoter; and(ii) a second expression construct comprising a nucleic acid encoding apolypeptide comprising a V_(L) operably linked to a promoter.

The present disclosure also provides an isolated or recombinant cellexpressing a protein of the disclosure.

In one example, the cell comprises the expression construct of thedisclosure or:

(i) a first expression construct comprising a nucleic acid encoding apolypeptide comprising a V_(H) operably linked to a promoter; and(ii) a second expression construct comprising a nucleic acid encoding apolypeptide comprising a V_(L) operably linked to a promoter.

Examples of cells of the present disclosure include bacterial cells,yeast cells, insect cells or mammalian cells.

The present disclosure additionally provides methods for producing aprotein or antibody of the disclosure. For example, such a methodinvolves maintaining the expression construct(s) of the disclosure underconditions sufficient for the protein to be produced.

In one example, a method for producing a protein or antibody of thedisclosure comprises culturing the cell of the disclosure underconditions sufficient for the protein or antibody to be produced and,optionally, secreted.

In one example, the method for producing a protein of the disclosureadditionally comprises isolating the protein or antibody and,optionally, formulating the protein or antibody into a pharmaceuticalcomposition.

The present disclosure additionally provides a composition comprising aprotein or antibody of the disclosure and a pharmaceutically acceptablecarrier.

The present disclosure additionally provides a composition comprising:

(i) an antibody comprising a heavy chain comprising an amino acidsequence set forth in SEQ ID NO: 64 and a light chain comprising anamino acid sequence set forth in SEQ ID NO: 65; and(ii) (a) an antibody comprising a heavy chain comprising an amino acidsequence set forth in SEQ ID NO: 64 and a light chain comprising anamino acid sequence set forth in SEQ ID NO: 65; and/or

-   -   (b) an antibody comprising one heavy chain comprising an amino        acid sequence set forth in SEQ ID NO: 64 and one heavy chain        comprising an amino acid sequence set forth in SEQ ID NO: 68 and        two light chains comprising an amino acid sequence set forth in        SEQ ID NO: 65, and, optionally, a pharmaceutically acceptable        carrier.

The present disclosure also provides a method for treating or preventinga G-CSF-mediated condition in a subject, the method comprisingadministering the protein, antibody or composition of the disclosure. Inthis regard, a protein, antibody or composition can be used to prevent arelapse of a condition, and this is considered preventing the condition.

In one example, the G-CSF-mediated condition is an autoimmune disease,an inflammatory disease or cancer. For example, the autoimmune diseaseor the inflammatory disease is arthritis, multiple sclerosis, pulmonaryinflammation or chronic obstructive pulmonary disease.

In one example, the method comprises administering an amount of theprotein or antibody sufficient to reduce the number of neutrophils in asubject without inducing neutropenia.

The present disclosure alternatively or additionally provides a methodfor reducing the number of neutrophils in a subject without inducingneutropenia, the method comprising administering a protein comprising anantigen binding site of an antibody that binds (or specifically binds)to hG-CSFR to the subject. An exemplary protein is an antibody orcomprises an antigen binding domain thereof (e.g., a V_(H) and/or aV_(L)) or is an antigen binding fragment thereof. Exemplary proteins andantibodies are described herein.

In one example, a method described herein comprises administering anamount of the protein or antibody sufficient to reduce the number ofneutrophils in a subject without inducing moderate neutropenia.

In one example, a method described herein comprises administering anamount of the protein or antibody sufficient to reduce the number ofneutrophils in a subject without inducing severe neutropenia.

In one example, a method described herein comprises administeringbetween about 0.05 mg/kg and 30 mg/kg of the protein or antibody. Forexample, the method comprising administering between 0.1 mg/kg and 10mg/kg or between 0.2 mg/kg and 5 mg/kg of the protein or antibody. Inone example, the method comprises administering about 0.5-2.0 mg/kg ofthe protein or antibody.

The present disclosure also provides for use of a protein or antibody asdescribed herein in any example in medicine.

The present disclosure also provides for use of a protein or antibody asdescribed herein according to any example in the manufacture of amedicament to treat a G-CSF-mediated condition.

The present disclosure also provides a method for localizing and/ordetecting and/or diagnosing and/or prognosing G-CSF-mediated conditionassociated with a cell expressing G-CSFR, the method comprisingdetecting in vivo a protein or antibody as described herein bound to theG-CSFR expressing cell, if present, wherein the protein or antibody isconjugated to a detectable tag.

In one example, the method additionally comprises administering theprotein to the subject.

The present disclosure also provides a method for detecting G-CSFR or acell expressing same in a sample, the method comprising contacting thesample with a protein or antibody as described herein according to anyexample such that a complex forms and detecting the complex, whereindetection of the complex is indicative of G-CSFR or a cell expressingsame in the sample.

The present disclosure also provides a method for diagnosing orprognosing a G-CSF-mediated condition, the method comprising performinga method as described herein according to any example to detect G-CSFRor a cell expressing same, wherein detection of the G-CSFR or cellexpressing same is diagnostic or prognostic of the condition.

The present disclosure also provides a kit comprising a protein orantibody as described herein according to any example packaged withinstructions for use in a method as described herein.

Key to Sequence Listing

SEQ ID NO: 1—amino acids 25-335 of Homo sapiens G-CSFR (hG-CSFR) with aC-terminal polyhistidine tag

SEQ ID NO: 2—V_(H) of C1.2 SEQ ID NO: 3—V_(L) of C1.2 SEQ ID NO: 4—V_(H)of C1.2G SEQ ID NO: 5—V_(L) of C1.2G SEQ ID NO: 6—HCDR1 of C1.2 SEQ IDNO: 7—HCDR2 of C1.2 SEQ ID NO: 8—HCDR3 of C1.2 SEQ ID NO: 9—LCDR1 ofC1.2 SEQ ID NO: 10—LCDR2 of C1.2 SEQ ID NO: 11—LCDR3 of C1.2

SEQ ID NO: 12—consensus sequence of HCDR3 of C1.2SEQ ID NO: 13—consensus sequence of LCDR3 of C1.2SEQ ID NO: 14—V_(L) of antibody 987SEQ ID NO: 15—V_(H) of antibody 987SEQ ID NO: 16—V_(L) of antibody 95SEQ ID NO: 17—V_(L) of antibody 79SEQ ID NO: 18—V_(L) of antibody 83SEQ ID NO: 19—V_(L) of antibody 1003SEQ ID NO: 20—V_(H) of antibody 1003SEQ ID NO: 21—V_(L) of antibody 44SEQ ID NO: 22—V_(L) of antibody 97SEQ ID NO: 23—V_(L) of antibody 986SEQ ID NO: 24—V_(H) of antibody 986SEQ ID NO: 25—V_(L) of antibody 56SEQ ID NO: 26—V_(L) of antibody 77SEQ ID NO: 27—V_(L) of antibody 54SEQ ID NO: 28—V_(H) of antibody 54SEQ ID NO: 29—V_(L) of antibody 802SEQ ID NO: 30—V_(L) of antibody 967SEQ ID NO: 31—V_(H) of antibody 967SEQ ID NO: 32—V_(L) of antibody 989SEQ ID NO: 33—V_(H) of antibody 989SEQ ID NO: 34—V_(L) of antibody 63SEQ ID NO: 35—V_(L) of antibody 1002SEQ ID NO: 36—V_(H) of antibody 1002SEQ ID NO: 37—V_(L) of antibody 994SEQ ID NO: 38—V_(H) of antibody 994SEQ ID NO: 39—V_(L) of antibody 969SEQ ID NO: 40—V_(H) of antibody 969SEQ ID NO: 41—V_(L) of antibody 1000SEQ ID NO: 42—V_(H) of antibody 1000SEQ ID NO: 43—V_(L) of antibody 94SEQ ID NO: 44—V_(L) of antibody 975SEQ ID NO: 45—V_(H) of antibody 975SEQ ID NO: 46—V_(L) of antibody 75SEQ ID NO: 47—V_(L) of antibody 814SEQ ID NO: 48—V_(L) of antibody 973SEQ ID NO: 49—V_(H) of antibody 973SEQ ID NO: 50—V_(L) of antibody 977SEQ ID NO: 51—V_(H) of antibody 977SEQ ID NO: 52—V_(L) of antibody 984SEQ ID NO: 53—V_(H) of antibody 984SEQ ID NO: 54—V_(L) of antibody 61SEQ ID NO: 55—V_(H) of antibody 852SEQ ID NO: 56—V_(L) of antibody 996SEQ ID NO: 57—V_(H) of antibody 996SEQ ID NO: 58—V_(L) of antibody 43SEQ ID NO: 59—V_(H) of antibody 43SEQ ID NO: 60—V_(L) of antibody 999SEQ ID NO: 61—V_(H) of antibody 999SEQ ID NO: 62—V_(L) of antibody 870SEQ ID NO: 63—V_(L) of antibody 877SEQ ID NO: 64—Heavy chain of C1.2G with stabilized IgG4 constant regionSEQ ID NO: 65—Light chain of C1.2G with kappa constant regionSEQ ID NO: 66—sequence of exemplary h-GCSFRSEQ ID NO: 67—polypeptide comprising Ig and CRH domains of Macacafascicularis G-CSFR (cynoG-CSFR) with a C-terminal polyhistidine tagSEQ ID NO: 68—Heavy chain of C1.2G with stabilized IgG4 constant regionand lacking C-terminal lysine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation showing inhibition ofG-CSF-mediated proliferation of BaF3 cells by increasing concentrationsof various anti-G-CSFR antibodies. The relative IC₅₀ values for eachantibody were; 10.1 μg/mL for mAb711, 37.4 μg/ml for mAb 774, 0.8 μg/mLfor C1.2G and was not determinable for mAb 744.

FIG. 2A is a graphical representation showing the relative binding ofC1.2G and mAb744 to a series of alanine point mutants of SEQ ID NO: 1compared to their binding to SEQ ID NO: 1 (positions of mutations areindicated with reference to SEQ ID NO: 1). The fold decrease in K_(D) ofthe antibody for the mutant receptor compared to SEQ ID NO: 1 isdepicted.

FIG. 2B is a graphical representation showing the relative binding ofC1.2G, mAb744 and mAb774 to a series of alanine point mutants of SEQ IDNO: 1 compared to their binding to SEQ ID NO: 1 (positions of mutationsare indicated with reference to SEQ ID NO: 1). The fold decrease inK_(D) of the antibody for the mutant receptor compared to SEQ ID NO: 1is depicted.

FIG. 3 is a graphical representation showing results of an assay inwhich pegylated G-CSF was administered to cynomolgus monkeys and one daylater C1.2 was administered. The number of neutrophils per μL blood wasassessed.

DETAILED DESCRIPTION General

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or groups of compositionsof matter.

Those skilled in the art will appreciate that the present disclosure issusceptible to variations and modifications other than thosespecifically described. It is to be understood that the disclosureincludes all such variations and modifications. The disclosure alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to applymutatis mutandis to any other example of the disclosure unlessspecifically stated otherwise.

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (for example, in cellculture, molecular genetics, immunology, immunohistochemistry, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present disclosure are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The description and definitions of variable regions and parts thereof,immunoglobulins, antibodies and fragments thereof herein may be furtherclarified by the discussion in Kabat Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia andLesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883,1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

Selected Definitions

For the purposes of nomenclature only and not limitation an exemplarysequence of a human G-CSFR is set out in NCBI Reference Sequence:NP_(—)000751.1 (and set out in SEQ ID NO: 66). The sequence ofcynomolgus monkey G-CSFR can be determined using sequences providedherein and/or in publically available databases and/or determined usingstandard techniques (e.g., as described in Ausubel et al., (editors),Current Protocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present) orSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (1989)) Reference to human G-CSFR may beabbreviated to hG-CSFR and reference to cynomolgus monkey G-CSFR may beabbreviated to cynoG-CSFR. Reference to soluble G-CSFR refers topolypeptides comprising the ligand binding region of G-CSFR. The Ig andCRH domains of the G-CSFR are involved in ligand binding and receptordimerization (Layton et al., J. Biol Chem., 272: 29735-29741, 1997 andFukunaga et al, EMBO J. 10: 2855-2865, 1991). Soluble forms of G-CSFRcomprising these portions of the receptor have been used in variousstudies of the receptor and mutation of the free cysteines at positions78, 163, and 228 of the receptor assists in expression and isolation ofthe soluble receptor polypeptide (Mine et al., Biochem., 43: 2458-24642004) without affecting ligand binding. In the present studies solubleforms of the receptor comprising amino acids 25-335 of hG-CSFR withmutations C78A, C163S and C228S were used (e.g. SEQ ID NO:1) and thecorresponding segment of cynoG-CSFR with the cysteine mutations was used(e.g., SEQ ID NO 67) for studies on the cynomolgus monkey receptor.Various point mutations of the soluble receptor of SEQ ID NO:1 and SEQID NO: 67 have also been utilized. Reference to hG-CSFR-Fc means thepolypeptide of SEQ ID NO:1 wherein the C-terminal polyhistidine tag hasbeen replaced with an Fc sequence (e.g., a polypeptide comprising aminoacids 1-311 of SEQ ID NO: 1 fused to an Fc). cynoG-CSFR-Fc means thecorresponding segment of cynoG-CSFR with an Fc sequence attached to itsC-terminus (e.g., a polypeptide comprising amino acids 1-311 of SEQ IDNO: 67 fused to an Fc). The inventors have shown that antibodies andproteins comprising antigen binding sites thereof (e.g., Fab) bind towild type hG-CSF polypeptides and to these mutant proteins with highlysimilar affinity. Accordingly, studies using the mutant proteins are amodel of studies using hG-CSFR and/or cynoG-CSFR.

Reference herein to G-CSF includes native forms of G-CSF, mutant formsthereof, e.g., filgrastim and pegylated forms of G-CSF or filgrastim.This term also encompasses mutant forms of G-CSF retaining activity tobind to G-CSFR (e.g., hG-CSFR) and induce signaling.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally-associated components that accompany it in itsnative state; is substantially free of other proteins from the samesource. A protein may be rendered substantially free of naturallyassociated components or substantially purified by isolation, usingprotein purification techniques known in the art. By “substantiallypurified” is meant the protein is substantially free of contaminatingagents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or96% or 97% or 98% or 99% free of contaminating agents.

The term “recombinant” shall be understood to mean the product ofartificial genetic recombination. Accordingly, in the context of arecombinant protein comprising an antibody antigen binding domain, thisterm does not encompass an antibody naturally-occurring within asubject's body that is the product of natural recombination that occursduring B cell maturation. However, if such an antibody is isolated, itis to be considered an isolated protein comprising an antibody antigenbinding domain. Similarly, if nucleic acid encoding the protein isisolated and expressed using recombinant means, the resulting protein isa recombinant protein comprising an antibody antigen binding domain. Arecombinant protein also encompasses a protein expressed by artificialrecombinant means when it is within a cell, tissue or subject, e.g., inwhich it is expressed.

The term “protein” shall be taken to include a single polypeptide chain,i.e., a series of contiguous amino acids linked by peptide bonds or aseries of polypeptide chains covalently or non-covalently linked to oneanother (i.e., a polypeptide complex). For example, the series ofpolypeptide chains can be covalently linked using a suitable chemical ora disulphide bond. Examples of non-covalent bonds include hydrogenbonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term “polypeptide” or “polypeptide chain” will be understood fromthe foregoing paragraph to mean a series of contiguous amino acidslinked by peptide bonds.

As used herein, the term “antigen binding site” shall be taken to mean astructure formed by a protein that is capable of binding or specificallybinding to an antigen. The antigen binding site need not be a series ofcontiguous amino acids, or even amino acids in a single polypeptidechain. For example, in a Fv produced from two different polypeptidechains the antigen binding site is made up of a series of amino acids ofa V_(L) and a V_(H) that interact with the antigen and that aregenerally, however not always in the one or more of the CDRs in eachvariable region. In some examples, an antigen binding site is a V_(H) ora V_(L) or a Fv.

The skilled artisan will be aware that an “antibody” is generallyconsidered to be a protein that comprises a variable region made up of aplurality of polypeptide chains, e.g., a polypeptide comprising a V_(L)and a polypeptide comprising a V_(H). An antibody also generallycomprises constant domains, some of which can be arranged into aconstant region, which includes a constant fragment or fragmentcrystallizable (Fc), in the case of a heavy chain. A V_(H) and a V_(L)interact to form a Fv comprising an antigen binding region that iscapable of specifically binding to one or a few closely relatedantigens. Generally, a light chain from mammals is either a κ lightchain or a λ light chain and a heavy chain from mammals is α, δ, ε, γ,or μ. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, andIgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass.The term “antibody” also encompasses humanized antibodies, primatizedantibodies, human antibodies and chimeric antibodies.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antigen binding fragment of an antibody.Specifically, whole antibodies include those with heavy and light chainsincluding an Fc region. The constant domains may be wild-type sequenceconstant domains (e.g., human wild-type sequence constant domains) oramino acid sequence variants thereof.

As used herein, “variable region” refers to the portions of the lightand/or heavy chains of an antibody as defined herein that is capable ofspecifically binding to an antigen and includes amino acid sequences ofcomplementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3,and framework regions (FRs). Exemplary variable regions comprise threeor four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with threeCDRs. In the case of a protein derived from an IgNAR, the protein maylack a CDR2. V_(H) refers to the variable region of the heavy chain.V_(L) refers to the variable region of the light chain.

As used herein, the term “complementarity determining regions” (syn.CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues ofan antibody variable region the presence of which are necessary forantigen binding. Each variable region typically has three CDR regionsidentified as CDR1, CDR2 and CDR3. The amino acid positions assigned toCDRs and FRs can be defined according to Kabat Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,1987 and 1991 or other numbering systems in the performance of thisdisclosure, e.g., the canonical numbering system of Chothia and Lesk J.Mol Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989;and/or Al-Lazikani et al., J Mol Biol 273: 927-948, 1997; the IMGTnumbering system of Lefranc et al., Devel. And Compar. Immunol., 27:55-77, 2003; or the AHO numbering system of Honnegher and Pliikthun J.Mol. Biol., 309: 657-670, 2001. For example, according to the numberingsystem of Kabat, V_(H) framework regions (FRs) and CDRs are positionedas follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65(CDR2), 66-94 (FR3), 95-102 (CDR3) and 103-113 (FR4). According to thenumbering system of Kabat, V_(L) FRs and CDRs are positioned as follows:residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88(FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is notlimited to FRs and CDRs as defined by the Kabat numbering system, butincludes all numbering systems, including those discussed above. In oneexample, reference herein to a CDR (or a FR) is in respect of thoseregions according to the Kabat numbering system.

“Framework regions” (FRs) are those variable region residues other thanthe CDR residues.

As used herein, the term “Fv” shall be taken to mean any protein,whether comprised of multiple polypeptides or a single polypeptide, inwhich a V_(L) and a V_(H) associate and form a complex having an antigenbinding site, i.e., capable of specifically binding to an antigen. TheV_(H) and the V_(L) which form the antigen binding site can be in asingle polypeptide chain or in different polypeptide chains.Furthermore, an Fv of the disclosure (as well as any protein of thedisclosure) may have multiple antigen binding sites which may or may notbind the same antigen. This term shall be understood to encompassfragments directly derived from an antibody as well as proteinscorresponding to such a fragment produced using recombinant means. Insome examples, the V_(H) is not linked to a heavy chain constant domain(C_(H)) 1 and/or the V_(L) is not linked to a light chain constantdomain (C_(L)). Exemplary Fv containing polypeptides or proteins includea Fab fragment, a Fab′ fragment, a F(ab′) fragment, a scFv, a diabody, atriabody, a tetrabody or higher order complex, or any of the foregoinglinked to a constant region or domain thereof, e.g., C_(H)2 or C_(H)3domain, e.g., a minibody. A “Fab fragment” consists of a monovalentantigen-binding fragment of an immunoglobulin, and can be produced bydigestion of a whole antibody with the enzyme papain, to yield afragment consisting of an intact light chain and a portion of a heavychain or can be produced using recombinant means. A “Fab′ fragment” ofan antibody can be obtained by treating a whole antibody with pepsin,followed by reduction, to yield a molecule consisting of an intact lightchain and a portion of a heavy chain comprising a V_(H) and a singleconstant domain. Two Fab′ fragments are obtained per antibody treated inthis manner. A Fab′ fragment can also be produced by recombinant means.A “F(ab′)2 fragment” of an antibody consists of a dimer of two Fab′fragments held together by two disulfide bonds, and is obtained bytreating a whole antibody molecule with the enzyme pepsin, withoutsubsequent reduction. A “Fab₂” fragment is a recombinant fragmentcomprising two Fab fragments linked using, for example a leucine zipperor a C_(H)3 domain. A “single chain Fv” or “scFv” is a recombinantmolecule containing the variable region fragment (Fv) of an antibody inwhich the variable region of the light chain and the variable region ofthe heavy chain are covalently linked by a suitable, flexiblepolypeptide linker.

As used herein, the term “binds” in reference to the interaction of aprotein or an antigen binding site thereof with an antigen means thatthe interaction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the antigen. For example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody binds to epitope “A”, thepresence of a molecule containing epitope “A” (or free, unlabeled “A”),in a reaction containing labeled “A” and the protein, will reduce theamount of labeled “A” bound to the antibody.

As used herein, the term “specifically binds” or “binds specifically”shall be taken to mean that a protein of the disclosure reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular antigen or cell expressing samethan it does with alternative antigens or cells. For example, a proteinbinds to G-CSFR (e.g., hG-CSFR) with materially greater affinity (e.g.,20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200fold) than it does to other cytokine receptor or to antigens commonlyrecognized by polyreactive natural antibodies (i.e., by naturallyoccurring antibodies known to bind a variety of antigens naturally foundin humans). In an example of the present disclosure, a protein that“specifically binds” to one form of hG-CSFR or a polypeptide comprisinga region thereof (e.g., the ligand binding domain of hG-GCSFR) or apolypeptide comprising amino acids 1-311 of SEQ ID NO: 1 with anaffinity at least 20 fold or 40 fold or 60 fold or 80 fold or 100 foldor 150 fold or 200 fold greater than it does to a mutant form of hG-CSFRor a polypeptide comprising a region thereof (e.g., a mutant form of theligand binding domain of h-GCSFR) or a mutant form of SEQ ID NO: 1comprising an alanine substituted for the native arginine at position287. Additional exemplary changes to SEQ ID NO: 1 and their effect onbinding are described herein. Generally, but not necessarily, referenceto binding means specific binding, and each term shall be understood toprovide explicit support for the other term.

As used herein, the term “does not detectably bind” shall be understoodto mean that a protein, e.g., an antibody, binds to a candidate antigenat a level less than 10%, or 8% or 6% or 5% above background. Thebackground can be the level of binding signal detected in the absence ofthe protein and/or in the presence of a negative control protein (e.g.,an isotype control antibody) and/or the level of binding detected in thepresence of a negative control antigen. The level of binding is detectedusing biosensor analysis (e.g. Biacore) in which the protein isimmobilized and contacted with an antigen.

As used herein, the term “does not significantly bind” shall beunderstood to mean that the level of binding of a protein of thedisclosure to a polypeptide is not statistically significantly higherthan background, e.g., the level of binding signal detected in theabsence of the protein and/or in the presence of a negative controlprotein (e.g., an isotype control antibody) and/or the level of bindingdetected in the presence of a negative control polypeptide. The level ofbinding is detected using biosensor analysis (e.g. Biacore) in which theprotein is immobilized and contacted with an antigen.

As used herein, phrases referring to “reduced binding” or “binding beingat a lower level” in relation to an antigen will be understood to meanthat an antibody binds to an antigen (e.g., an alanine point mutant ofSEQ ID NO:1 at any one of positions 287, 237, 198, 172, 171 or 111) withan affinity at least about 20 fold or 40 fold or 60 fold less than acontrol epitope or antigen (e.g. SEQ ID NO:1). For example, a protein ofthe present disclosure can bind to a polypeptide of SEQ ID NO: 1 inwhich an alanine is substituted for the histidine at position 237 at alevel 20 fold or 40 fold or 60 fold less than it binds to a polypeptideof SEQ ID NO: 1. Preferably, the protein binds at a level 20 fold less,more preferably 40 fold less, still more preferably 60 fold less.

A protein or antibody may be considered to “preferentially bind” to apolypeptide if it binds that polypeptide with a dissociation constant(K_(D)) that is less than the protein's or antibody's K_(D) for anotherpolypeptide. In one example, a protein or antibody is considered topreferentially bind to a polypeptide if it binds the polypeptide with anaffinity (i.e., K_(D)) that is at least about 20 fold or 40 fold or 60fold or 80 fold or 100 fold or 120 fold or 140 fold or 160 fold morethan the protein's or antibody's K_(D) for another polypeptide.

As used herein, the term “similar affinity” will be understood to meanthat a protein of the present disclosure binds to two antigens (e.g.,the ligand binding domain of G-CSFR from humans and from cynomolgusmonkeys) with affinities that are within about 5 fold or less of oneanother, e.g., within about 4, 3, 2, or 1 fold of one another, such as,within about 0.5 fold of one another or the levels of binding aresubstantially identical, e.g., when the affinity is assessed byimmobilizing the two antigens (e.g., the ligand binding domain of G-CSFRor extracellular domains from humans and from cynomolgus monkeys) andcontacting the immobilized proteins with a protein of the disclosure.

For the purposes of clarification and as will be apparent to the skilledartisan based on the exemplified subject matter herein, reference to“affinity” in this specification is a reference to K_(D) of a protein orantibody.

For the purposes of clarification and as will be apparent to the skilledartisan based on the description herein, reference to an “affinity of atleast about” will be understood to mean that the affinity (or K_(D)) isequal to the recited value or higher (i.e., the value recited as theaffinity is lower), i.e., an affinity of 2 nM is greater than anaffinity of 3 nM. Stated another way, this term could be “an affinity ofX or less”, wherein X is a value recited herein.

An “IC₅₀ of at least about” will be understood to mean that the IC₅₀ isequal to the recited value or greater (i.e., the value recited as theIC₅₀ is lower), i.e., an IC₅₀ of 2 nM is greater than an IC₅₀ of 3 nM.Stated another way, this term could be “an IC₅₀ of X or less”, wherein Xis a value recited herein.

As used herein, the term “epitope” (syn. “antigenic determinant”) shallbe understood to mean a region of hG-CSFR to which a protein comprisingan antigen binding site of an antibody binds. This term is notnecessarily limited to the specific residues or structure to which theprotein makes contact. For example, this term includes the regionspanning amino acids contacted by the protein and/or 5-10 or 2-5 or 1-3amino acids outside of this region. In some examples, the epitopecomprises a series of discontinuous amino acids that are positionedclose to one another when hG-CSFR is folded, i.e., a “conformationalepitope”. For example, a conformational epitope comprises amino acids inone or more or two or more or all of the regions corresponding to111-115, 170-176, 218-234 and/or 286-300 of SEQ ID NO: 1. The skilledartisan will also be aware that the term “epitope” is not limited topeptides or polypeptides. For example, the term “epitope” includeschemically active surface groupings of molecules such as sugar sidechains, phosphoryl side chains, or sulfonyl side chains, and, in certainexamples, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics.

The term “competitively inhibits” shall be understood to mean that aprotein of the disclosure (or an antigen binding site thereof) reducesor prevents binding of a recited antibody or protein to G-CSFR, e.g., tohG-CSFR. This may be due to the protein (or antigen binding site) andantibody binding to the same or an overlapping epitope. It will beapparent from the foregoing that the protein need not completely inhibitbinding of the antibody, rather it need only reduce binding by astatistically significant amount, for example, by at least about 10% or20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Preferably,the protein reduces binding of the antibody by at least about 30%, morepreferably by at least about 50%, more preferably, by at least about70%, still more preferably by at least about 75%, even more preferably,by at least about 80% or 85% and even more preferably, by at least about90%. Methods for determining competitive inhibition of binding are knownin the art and/or described herein. For example, the antibody is exposedto G-CSFR either in the presence or absence of the protein. If lessantibody binds in the presence of the protein than in the absence of theprotein, the protein is considered to competitively inhibit binding ofthe antibody. In one example, the competitive inhibition is not due tosteric hindrance.

“Overlapping” in the context of two epitopes shall be taken to mean thattwo epitopes share a sufficient number of amino acid residues to permita protein (or antigen binding site thereof) that binds to one epitope tocompetitively inhibit the binding of a protein (or antigen binding site)that binds to the other epitope. For example, the “overlapping” epitopesshare at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 aminoacids.

As used herein, the term “neutralize” shall be taken to mean that aprotein is capable of blocking, reducing or preventing G-CSF-mediatedsignaling in a cell through the G-CSFR. Methods for determiningneutralization are known in the art and/or described herein.

As used herein, the term “condition” refers to a disruption of orinterference with normal function, and is not to be limited to anyspecific condition, and will include diseases or disorders.

As used herein, a “G-CSF-associated condition” refers to any conditionthat is caused by or associated with neutrophils, an excess of G-CSF orcells expressing G-CSFR or with administration of G-CSF. The skilledartisan will be readily able to determine such conditions. In thisregard, in some examples the condition is an inflammatory condition, anautoimmune condition or cancer (including metastasis).

As used herein, the terms “preventing”, “prevent” or “prevention”include administering a protein of the disclosure to thereby stop orhinder the development of at least one symptom of a condition. This termalso encompasses treatment of a subject in remission to prevent orhinder relapse. For example, a subject suffering fromrelapsing-remitting multiple sclerosis is treated during remission tothereby prevent a relapse.

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a protein described herein to thereby reduce or eliminateat least one symptom of a specified disease or condition.

As used herein, the term “neutropenia” will be understood to encompassmild neutropenia (1000<=ANC<1500), moderate neutropenia (500<=ANC<1000)and Severe neutropenia (ANC<500) (absolute neutrophil count (ANC)measured in cells per microliter of blood).

As used herein, the term “subject” shall be taken to mean any animalincluding humans, for example a mammal. Exemplary subjects include butare not limited to humans and non-human primates. For example, thesubject is a human.

Antibodies

In one example, a protein as described herein according to any exampleis an antibody.

Methods for generating antibodies are known in the art and/or describedin Harlow and Lane (editors) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, (1988). Generally, in such methods G-CSFR(e.g., hG-CSFR) or a region thereof (e.g., an extracellular domain) orimmunogenic fragment or epitope thereof or a cell expressing anddisplaying same (i.e., an immunogen), optionally formulated with anysuitable or desired carrier, adjuvant, or pharmaceutically acceptableexcipient, is administered to a non-human animal, for example, a mouse,chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. Theimmunogen may be administered intranasally, intramuscularly,sub-cutaneously, intravenously, intradermally, intraperitoneally, or byother known route.

The production of polyclonal antibodies may be monitored by samplingblood of the immunized animal at various points following immunization.One or more further immunizations may be given, if required to achieve adesired antibody titer. The process of boosting and titering is repeateduntil a suitable titer is achieved. When a desired level ofimmunogenicity is obtained, the immunized animal is bled and the serumisolated and stored, and/or the animal is used to generate monoclonalantibodies (mabs).

Monoclonal antibodies are one exemplary form of antibody contemplated bythe present disclosure. The term “monoclonal antibody” or “mAb” refersto a homogeneous antibody population capable of binding to the sameantigen(s), for example, to the same epitope within the antigen. Thisterm is not intended to be limited as regards to the source of theantibody or the manner in which it is made.

For the production of mAbs any one of a number of known techniques maybe used, such as, for example, the procedure exemplified in U.S. Pat.No. 4,196,265 or Harlow and Lane (1988), supra.

For example, a suitable animal is immunized with an immunogen underconditions sufficient to stimulate antibody producing cells. Rodentssuch as rabbits, mice and rats are exemplary animals. Micegenetically-engineered to express human antibodies and, for example, donot express murine antibodies, can also be used to generate an antibodyof the present disclosure (e.g., as described in WO2002/066630).

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsies of spleens, tonsils or lymph nodes, or from a peripheral bloodsample. The B cells from the immunized animal are then fused with cellsof an immortal myeloma cell, generally derived from the same species asthe animal that was immunized with the immunogen.

Hybrids are amplified by culture in a selective medium comprising anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary agents are aminopterin, methotrexate andazaserine.

The amplified hybridomas are subjected to a functional selection forantibody specificity and/or titer, such as, for example, by flowcytometry and/or immunohistochemistry and/or immunoassay (e.g.radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay,dot immunoassay, and the like).

Alternatively, ABL-MYC technology (NeoClone, Madison Wis. 53713, USA) isused to produce cell lines secreting MAbs (e.g., as described inLargaespada et al, J. Immunol. Methods. 197: 85-95, 1996).

Antibodies can also be produced or isolated by screening a displaylibrary, e.g., a phage display library, e.g., as described in U.S. Pat.No. 6,300,064 and/or U.S. Pat. No. 5,885,793. For example, the presentinventors have isolated fully human antibodies from a phage displaylibrary.

As described herein, some proteins of the present disclosure that bindhG-CSFR cross-react with cynoG-CSFR and/or bind to some mutant forms ofhG-CSFR or polypeptides comprising regions of hG-CSFR that have beenmutated and/or not others and/or bind to a specific epitope withinhG-CSFR. These characteristics can be used in the generation of anantibody or a protein comprising a binding site thereof.

For example, a phage display library is screened with a polypeptidecomprising the ligand binding domain of hG-CSFR to identify proteinsthat bind thereto. Mutant forms of the ligand binding domain of hG-CSFR(e.g., an alanine point mutant of SEQ ID NO:1 at position 287) to whichthe protein is not to detectably bind are then used to removecross-reactive proteins and mutant forms of the ligand binding domain ofhG-CSFR or regions thereof (e.g., an alanine point mutant of SEQ ID NO:1at position 168) to which the protein is to bind are used to isolateproteins that are correctly cross-reactive. A screening process forimmunization of a non-human mammal can also be devised based on theforegoing.

In another example, a phage display library is screened or an animal isimmunized with a polypeptide comprising the ligand binding domain ofcynoG-CSFR and identified proteins and/or antibodies are screened toidentify those that are cross-reactive with hG-CSFR and/or the ligandbinding domain thereof.

In a further example, a G-CSFR or a ligand binding domain thereof(optionally a mutant form to which C1.2 or C1.2G binds) is contactedwith C1.2 or C1.2G. A phage display library is then brought into contactwith the G-CSFR or the ligand binding domain and phage expressingproteins that can compete with C1.2 or C1.2G for binding selected.

In a still further example, a chimeric protein comprising, e.g., a mouseG-CSFR in which an epitope of interest from a hG-CSFR is substituted forthe corresponding mouse sequence. This chimeric protein is then used toimmunize mice (which are less likely to induce an immune responseagainst the mouse protein) and/or to screen a phage display library. Theresulting antibodies/proteins are then screened to identify those thatbind to hG-CSFR (particularly at the epitope of interest) and not mouseG-CSFR.

The antibody of the present disclosure may be a synthetic antibody. Forexample, the antibody is a chimeric antibody, a humanized antibody, ahuman antibody or a de-immunized antibody.

Chimeric Antibodies

In one example, an antibody described herein is a chimeric antibody. Theterm “chimeric antibody” refers to antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular species(e.g., murine, such as mouse) or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species (e.g., primate, such as human) or belonging to anotherantibody class or subclass. Typically chimeric antibodies utilize rodentor rabbit variable regions and human constant regions, in order toproduce an antibody with predominantly human domains. Methods forproducing chimeric antibodies are described in, e.g., U.S. Pat. No.4,816,567; and U.S. Pat. No. 5,807,715.

Humanized and Human Antibodies

The antibodies of the present disclosure may be humanized or human.

The term “humanized antibody” shall be understood to refer to a subclassof chimeric antibodies having an antigen binding site or variable regionderived from an antibody from a non-human species and the remainingantibody structure based upon the structure and/or sequence of a humanantibody. In a humanized antibody, the antigen-binding site generallycomprises the complementarity determining regions (CDRs) from thenon-human antibody grafted onto appropriate FRs in the variable regionsof a human antibody and the remaining regions from a human antibody.Antigen binding sites may be wild-type (i.e., identical to those of thenon-human antibody) or modified by one or more amino acid substitutions.In some instances, FR residues of the human antibody are replaced bycorresponding non-human residues.

Methods for humanizing non-human antibodies or parts thereof (e.g.,variable regions) are known in the art. Humanization can be performedfollowing the method of U.S. Pat. No. 5,225,539, or U.S. Pat. No.5,585,089. Other methods for humanizing an antibody are not excluded.

The term “human antibody” as used herein refers to antibodies havingvariable regions (e.g. V_(H), V_(L)) and, optionally constant regionsderived from or corresponding to sequences found in humans, e.g. in thehuman germline or somatic cells. The “human” antibodies can includeamino acid residues not encoded by human sequences, e.g. mutationsintroduced by random or site directed mutations in vitro (in particularmutations which involve conservative substitutions or mutations in asmall number of residues of the antibody, e.g. in 1, 2, 3, 4, 5 or 6 ofthe residues of the antibody, e.g. in 1, 2, 3, 4, 5 or 6 of the residuesmaking up one or more of the CDRs of the antibody). These “humanantibodies” do not actually need to be produced by a human, rather, theycan be produced using recombinant means and/or isolated from atransgenic animal (e.g., mouse) comprising nucleic acid encoding humanantibody constant and/or variable regions (e.g., as described above).Human antibodies can be produced using various techniques known in theart, including phage display libraries (e.g., as described in U.S. Pat.No. 5,885,793).

Human antibodies which recognize a selected epitope can also begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (e.g., as described in U.S. Pat. No.5,565,332).

Exemplary human antibodies are described herein and include C1.2 andC1.2G and/or variable regions thereof. These human antibodies provide anadvantage of reduced immunogenicity in a human compared to non-humanantibodies.

Antibody Binding Domain Containing Proteins Single-Domain Antibodies

In some examples, a protein of the disclosure is or comprises asingle-domain antibody (which is used interchangeably with the term“domain antibody” or “dAb”). A single-domain antibody is a singlepolypeptide chain comprising all or a portion of the heavy chainvariable region of an antibody. In certain examples, a single-domainantibody is a human single-domain antibody (Domantis, Inc., Waltham,Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Diabodies, Triabodies, Tetrabodies

In some examples, a protein of the disclosure is or comprises a diabody,triabody, tetrabody or higher order protein complex such as thosedescribed in WO98/044001 and/or WO94/007921.

For example, a diabody is a protein comprising two associatedpolypeptide chains, each polypeptide chain comprising the structureV_(L)-X-V_(H) or V_(H)-X-V_(L), wherein V_(L) is an antibody light chainvariable region, V_(H) is an antibody heavy chain variable region, X isa linker comprising insufficient residues to permit the V_(H) and V_(L)in a single polypeptide chain to associate (or form an Fv) or is absent,and wherein the V_(H) of one polypeptide chain binds to a V_(L) of theother polypeptide chain to form an antigen binding site, i.e., to form aFv molecule capable of specifically binding to one or more antigens. TheV_(L) and V_(H) can be the same in each polypeptide chain or the V_(L)and V_(H) can be different in each polypeptide chain so as to form abispecific diabody (i.e., comprising two Fvs having differentspecificity).

Single Chain Fv (scFv)

The skilled artisan will be aware that scFvs comprise V_(H) and V_(L)regions in a single polypeptide chain and a polypeptide linker betweenthe V_(H) and V_(L) which enables the scFv to form the desired structurefor antigen binding (i.e., for the V_(H) and V_(L) of the singlepolypeptide chain to associate with one another to form a Fv). Forexample, the linker comprises in excess of 12 amino acid residues with(Gly₄Ser)₃ being one of the more favored linkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (ordiFv or dsFv), in which a single cysteine residue is introduced into aFR of V_(H) and a FR of V_(L) and the cysteine residues linked by adisulfide bond to yield a stable Fv.

Alternatively, or in addition, the present disclosure encompasses adimeric scFv, i.e., a protein comprising two scFv molecules linked by anon-covalent or covalent linkage, e.g., by a leucine zipper domain(e.g., derived from Fos or Jun). Alternatively, two scFvs are linked bya peptide linker of sufficient length to permit both scFvs to form andto bind to an antigen, e.g., as described in US20060263367.

Heavy Chain Antibodies

Heavy chain antibodies differ structurally from many other forms ofantibodies, in so far as they comprise a heavy chain, but do notcomprise a light chain. Accordingly, these antibodies are also referredto as “heavy chain only antibodies”. Heavy chain antibodies are foundin, for example, camelids and cartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chainantibodies are generally referred to as “V_(HH) domains” in camelidantibodies and V-NAR in IgNAR, in order to distinguish them from theheavy chain variable regions that are present in conventional 4-chainantibodies (which are referred to as “V_(H) domains”) and from the lightchain variable regions that are present in conventional 4-chainantibodies (which are referred to as “V_(L) domains”).

A general description of heavy chain antibodies from camelids and thevariable regions thereof and methods for their production and/orisolation and/or use is found inter alia in the following referencesWO94/04678, WO97/49805 and WO 97/49805.

A general description of heavy chain antibodies from cartilaginous fishand the variable regions thereof and methods for their production and/orisolation and/or use is found inter alia in WO2005/118629.

Other Antibodies and Antibody Fragments

The present disclosure also contemplates other antibodies and antibodyfragments, such as:

(i) “key and hole” bispecific proteins as described in U.S. Pat. No.5,731,168;(ii) heteroconjugate proteins, e.g., as described in U.S. Pat. No.4,676,980;(iii) heteroconjugate proteins produced using a chemical cross-linker,e.g., as described in U.S. Pat. No. 4,676,980; and(iv) Fab₃ (e.g., as described in EP19930302894).

De-Immunized Antibodies and Proteins

The present disclosure also contemplates a de-immunized antibody orprotein. De-immunized antibodies and proteins have one or more epitopes,e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) tothereby reduce the likelihood that a mammal will raise an immuneresponse against the antibody or protein. Methods for producingde-immunized antibodies and proteins are known in the art and described,for example, in WO00/34317, WO2004/108158 and WO2004/064724.

Methods for introducing suitable mutations and expressing and assayingthe resulting protein will be apparent to the skilled artisan based onthe description herein.

Mutations to Proteins

The present disclosure also contemplates mutant forms of a protein ofthe disclosure. In this regard, data presented herein indicate siteswithin a CDR of a protein of the disclosure that can be changed inaddition to exemplary changes that can be made. The skilled person willunderstand that changes can additionally or alternatively be made withina FR of a variable region containing protein without inhibiting orsignificantly reducing its function in the context of the presentdisclosure.

For example, such a mutant protein comprises one or more conservativeamino acid substitutions compared to a sequence set forth herein. Insome examples, the protein comprises 30 or fewer or 20 or fewer or 10 orfewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 conservative aminoacid substitutions. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain and/or hydropathicity and/or hydrophilicity.

In one example, a mutant protein has only, or not more than, one or twoor three or four or five or six conservative amino acid changes whencompared to a naturally occurring protein. Details of conservative aminoacid changes are provided below. As the skilled person would be aware,e.g., from the disclosure herein, such minor changes can reasonably bepredicted not to alter the activity of the protein.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), (β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

The present disclosure also contemplates non-conservative amino acidchanges (e.g., substitutions) in a protein of the present disclosure,e.g., in a CDR, such as CDR3. For example, the present inventors haveidentified several non-conservative amino acid substitutions that can bemade while retaining an activity of a protein of the disclosure. In oneexample, the protein comprises fewer than 6 or 5 or 4 or 3 or 2 or 1non-conservative amino acid substitutions, e.g., in a CDR3, such as in aCDR3.

The present disclosure also contemplates one or more insertions ordeletions compared to a sequence set forth herein. In some examples, theprotein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or2 insertions and/or deletions.

Constant Regions

The present disclosure encompasses proteins and/or antibodies describedherein comprising a constant region of an antibody. This includesantigen binding fragments of an antibody fused to a Fc.

Sequences of constant regions useful for producing the proteins of thepresent disclosure may be obtained from a number of different sources.In some examples, the constant region or portion thereof of the proteinis derived from a human antibody. The constant region or portion thereofmay be derived from any antibody class, including IgM, IgG, IgD, IgA andIgE, and any antibody isotype, including IgG1, IgG2, IgG3 and IgG4. Inone example, the constant region is human isotype IgG4 or a stabilizedIgG4 constant region.

In one example, the Fc region of the constant region has a reducedability to induce effector function, e.g., compared to a native orwild-type human IgG1 or IgG3 Fc region. In one example, the effectorfunction is antibody-dependent cell-mediated cytotoxicity (ADCC) and/orantibody-dependent cell-mediated phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC). Methods for assessing the levelof effector function of an Fc region containing protein are known in theart and/or described herein.

In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4constant region), e.g., a human IgG4 Fc region. Sequences of suitableIgG4 Fc regions will be apparent to the skilled person and/or availablein publically available databases (e.g., available from National Centerfor Biotechnology Information).

In one example, the constant region is a stabilized IgG4 constantregion. The term “stabilized IgG4 constant region” will be understood tomean an IgG4 constant region that has been modified to reduce Fab armexchange or the propensity to undergo Fab arm exchange or formation of ahalf-antibody or a propensity to form a half antibody. “Fab armexchange” refers to a type of protein modification for human IgG4, inwhich an IgG4 heavy chain and attached light chain (half-molecule) isswapped for a heavy-light chain pair from another IgG4 molecule. Thus,IgG4 molecules may acquire two distinct Fab arms recognizing twodistinct antigens (resulting in bispecific molecules). Fab arm exchangeoccurs naturally in vivo and can be induced in vitro by purified bloodcells or reducing agents such as reduced glutathione. A “half antibody”forms when an IgG4 antibody dissociates to form two molecules eachcontaining a single heavy chain and a single light chain.

In one example, a stabilized IgG4 constant region comprises a proline atposition 241 of the hinge region according to the system of Kabat (Kabatet al., Sequences of Proteins of Immunological Interest Washington D.C.United States Department of Health and Human Services, 1987 and/or1991). This position corresponds to position 228 of the hinge regionaccording to the EU numbering system (Kabat et al., Sequences ofProteins of Immunological Interest Washington D.C. United StatesDepartment of Health and Human Services, 2001 and Edelman et al., Proc.Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue isgenerally a serine. Following substitution of the serine for proline,the IgG4 hinge region comprises a sequence CPPC. In this regard, theskilled person will be aware that the “hinge region” is a proline-richportion of an antibody heavy chain constant region that links the Fc andFab regions that confers mobility on the two Fab arms of an antibody.The hinge region includes cysteine residues which are involved ininter-heavy chain disulfide bonds. It is generally defined as stretchingfrom Glu226 to Pro243 of human IgG1 according to the numbering system ofKabat. Hinge regions of other IgG isotypes may be aligned with the IgG1sequence by placing the first and last cysteine residues forminginter-heavy chain disulphide (S—S) bonds in the same positions (see forexample WO2010/080538).

Additional examples of stabilized IgG4 antibodies are antibodies inwhich arginine at position 409 in a heavy chain constant region of humanIgG4 (according to the EU numbering system) is substituted with lysine,threonine, methionine, or leucine (e.g., as described in WO2006/033386).The Fc region of the constant region may additionally or alternativelycomprise a residue selected from the group consisting of: alanine,valine, glycine, isoleucine and leucine at the position corresponding to405 (according to the EU numbering system). Optionally, the hinge regioncomprises a proline at position 241 (i.e., a CPPC sequence) (asdescribed above).

In another example, the Fc region is a region modified to have reducedeffector function, i.e., a “non-immunostimulatory Fc region”. Forexample, the Fc region is an IgG1 Fc region comprising a substitution atone or more positions selected from the group consisting of 268, 309,330 and 331. In another example, the Fc region is an IgG1 Fc regioncomprising one or more of the following changes E233P, L234V, L235A anddeletion of G236 and/or one or more of the following changes A327G,A330S and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999;Shields et al., J Biol Chem. 276(9):6591-604, 2001). Additional examplesof non-immunostimulatory Fc regions are described, for example, inDall'Acqua et al., J Immunol. 177: 1129-1138 2006; and/or Hezareh JVirol; 75: 12161-12168, 2001).

In another example, the Fc region is a chimeric Fc region, e.g.,comprising at least one C_(H)2 domain from an IgG4 antibody and at leastone C_(H)3 domain from an IgG1 antibody, wherein the Fc region comprisesa substitution at one or more amino acid positions selected from thegroup consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399,409 and 427 (EU numbering) (e.g., as described in WO2010/085682).Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A,299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

Additional Modifications

The present disclosure also contemplates additional modifications to anantibody.

For example, the antibody comprises one or more amino acid substitutionsthat increase the half-life of the protein. For example, the antibodycomprises a Fc region comprising one or more amino acid substitutionsthat increase the affinity of the Fc region for the neonatal Fc region(FcRn). For example, the Fc region has increased affinity for FcRn atlower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in anendosome. In one example, the Fc region has increased affinity for FcRnat about pH 6 compared to its affinity at about pH 7.4, whichfacilitates the re-release of Fc into blood following cellularrecycling. These amino acid substitutions are useful for extending thehalf life of a protein, by reducing clearance from the blood.

Exemplary amino acid substitutions include T250Q and/or M428L or T252A,T254S and T266F or M252Y, S254T and T256E or H433K and N434F accordingto the EU numbering system. Additional or alternative amino acidsubstitutions are described, for example, in US20070135620 or U.S. Pat.No. 7,083,784.

Protein Production

In one example, a protein described herein according to any example isproduced by culturing a hybridoma under conditions sufficient to producethe protein, e.g., as described herein and/or as is known in the art.

Recombinant Expression

In another example, a protein described herein according to any exampleis recombinant.

In the case of a recombinant protein, nucleic acid encoding same can becloned into expression constructs or vectors, which are then transfectedinto host cells, such as E. coli cells, yeast cells, insect cells, ormammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO)cells, human embryonic kidney (HEK) cells, or myeloma cells that do nototherwise produce the protein. Exemplary cells used for expressing aprotein are CHO cells, myeloma cells or HEK cells. Molecular cloningtechniques to achieve these ends are known in the art and described, forexample in Ausubel et al., (editors), Current Protocols in MolecularBiology, Greene Pub. Associates and Wiley-Interscience (1988, includingall updates until present) or Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989). A widevariety of cloning and in vitro amplification methods are suitable forthe construction of recombinant nucleic acids. Methods of producingrecombinant antibodies are also known in the art, see, e.g., U.S. Pat.No. 4,816,567 or U.S. Pat. No. 5,530,101.

Following isolation, the nucleic acid is inserted operably linked to apromoter in an expression construct or expression vector for furthercloning (amplification of the DNA) or for expression in a cell-freesystem or in cells.

As used herein, the term “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences of agenomic gene, including the TATA box or initiator element, which isrequired for accurate transcription initiation, with or withoutadditional regulatory elements (e.g., upstream activating sequences,transcription factor binding sites, enhancers and silencers) that alterexpression of a nucleic acid, e.g., in response to a developmentaland/or external stimulus, or in a tissue specific manner. In the presentcontext, the term “promoter” is also used to describe a recombinant,synthetic or fusion nucleic acid, or derivative which confers, activatesor enhances the expression of a nucleic acid to which it is operablylinked. Exemplary promoters can contain additional copies of one or morespecific regulatory elements to further enhance expression and/or alterthe spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term “operably linked to” means positioning apromoter relative to a nucleic acid such that expression of the nucleicacid is controlled by the promoter.

Many vectors for expression in cells are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, a sequence encoding a protein (e.g.,derived from the information provided herein), an enhancer element, apromoter, and a transcription termination sequence. The skilled artisanwill be aware of suitable sequences for expression of a protein.Exemplary signal sequences include prokaryotic secretion signals (e.g.,pelB, alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II), yeast secretion signals (e.g., invertase leader, afactor leader, or acid phosphatase leader) or mammalian secretionsignals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirusimmediate early promoter (CMV-IE), human elongation factor 1-α promoter(EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chainpromoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter(RSV), Adenovirus major late promoter, β-actin promoter; hybridregulatory element comprising a CMV enhancer/β-actin promoter or animmunoglobulin promoter or active fragment thereof. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture; baby hamster kidney cells(BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as forexample a yeast cell selected from the group comprising Pichia pastoris,Saccharomyces cerevisiae and S. pombe, include, but are not limited to,the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or theTEF1 promoter.

Means for introducing the isolated nucleic acid or expression constructcomprising same into a cell for expression are known to those skilled inthe art. The technique used for a given cell depends on the knownsuccessful techniques. Means for introducing recombinant DNA into cellsinclude microinjection, transfection mediated by DEAE-dextran,transfection mediated by liposomes such as by using lipofectamine(Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNAuptake, electroporation and microparticle bombardment such as by usingDNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongstothers.

The host cells used to produce the protein may be cultured in a varietyof media, depending on the cell type used. Commercially available mediasuch as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing mammalian cells. Media for culturingother cell types discussed herein are known in the art.

Isolation of Proteins

Methods for isolating a protein are known in the art and/or describedherein.

Where a protein is secreted into culture medium, supernatants from suchexpression systems can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants. Alternatively, or additionally, supernatantscan be filtered and/or separated from cells expressing the protein,e.g., using continuous centrifugation.

The protein prepared from the cells can be purified using, for example,ion exchange, hydroxyapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, affinity chromatography(e.g., protein A affinity chromatography or protein G chromatography),or any combination of the foregoing. These methods are known in the artand described, for example in WO99/57134 or Ed Harlow and David Lane(editors) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, (1988).

The skilled artisan will also be aware that a protein can be modified toinclude a tag to facilitate purification or detection, e.g., apoly-histidine tag, e.g., a hexa-histidine tag, or a influenza virushemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, ora glutathione S-transferase (GST) tag. The resulting protein is thenpurified using methods known in the art, such as, affinity purification.For example, a protein comprising a hexa-his tag is purified bycontacting a sample comprising the protein with nickel-nitrilotriaceticacid (Ni-NTA) that specifically binds a hexa-his tag immobilized on asolid or semi-solid support, washing the sample to remove unboundprotein, and subsequently eluting the bound protein. Alternatively, orin addition a ligand or antibody that binds to a tag is used in anaffinity purification method.

Assaying Activity of a Protein Binding to G-CSFR and Mutants Thereof

It will be apparent to the skilled artisan from the disclosure hereinthat some proteins of the present disclosure bind to the ligand bindingdomain of hG-CSFR and to specific mutant forms of the ligand bindingdomain of hG-CSFR (e.g., SEQ ID NO: 1 without or with certain pointmutations) and/or bind to both human and cynomolgus monkey G-CSFR.Methods for assessing binding to a protein are known in the art, e.g.,as described in Scopes (In: Protein purification: principles andpractice, Third Edition, Springer Verlag, 1994). Such a method generallyinvolves labeling the protein and contacting it with immobilizedantigen. Following washing to remove non-specific bound protein, theamount of label and, as a consequence, bound protein is detected. Ofcourse, the protein can be immobilized and the antigen labeled.Panning-type assays can also be used. Alternatively, or additionally,surface plasmon resonance assays can be used.

The assays described above can also be used to detect the level ofbinding of a protein to hG-CSFR or a ligand binding domain thereof(e.g., SEQ ID NO: 1) or mutant form thereof.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thelysine at position 167 of SEQ ID NO: 1 and/or in which an alanine issubstituted for the histidine at position 168 of SEQ ID NO: 1 atsubstantially the same level (e.g., within 10% or 5% or 1%) as it bindsto SEQ ID NO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thearginine at position 287 of SEQ ID NO: 1 at a level at least about 100fold or 150 fold or 160 fold or 200 fold lower than it binds to apolypeptide of SEQ ID NO: 1. In one example, a protein of the presentdisclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for the arginine at position 287 of SEQ ID NO: 1 at a levelat least about 160 fold lower than it binds to a polypeptide of SEQ IDNO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thehistidine at position 237 of SEQ ID NO: 1 at a level at least about 20fold or 40 fold or 50 fold or 60 fold lower than it binds to apolypeptide of SEQ ID NO: 1. In one example, a protein of the presentdisclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for the histidine at position 237 of SEQ ID NO: 1 at a levelat least about 50 fold lower than it binds to a polypeptide of SEQ IDNO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for themethionine at position 198 of SEQ ID NO: 1 at a level at least about 20fold or 40 fold or 60 fold or 70 fold lower than it binds to apolypeptide of SEQ ID NO: 1. In one example, a protein of the presentdisclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for the methionine at position 198 of SEQ ID NO: 1 at alevel at least about 40 fold lower than it binds to a polypeptide of SEQID NO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thetyrosine at position 172 of SEQ ID NO: 1 at a level at least about 20fold or 30 fold or 40 fold lower than it binds to a polypeptide of SEQID NO: 1. In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thetyrosine at position 172 of SEQ ID NO: 1 at a level at least about 40fold lower than it binds to a polypeptide of SEQ ID NO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for theleucine at position 171 of SEQ ID NO: 1 at a level at least about 100fold or 120 fold or 130 fold or 140 fold lower than it binds to apolypeptide of SEQ ID NO: 1. In one example, a protein of the presentdisclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for the leucine at position 171 of SEQ ID NO: 1 at a levelat least about 140 fold lower than it binds to a polypeptide of SEQ IDNO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for theleucine at a position 111 of SEQ ID NO: 1 at a level at least about 20fold or 40 fold or 60 fold or 70 fold lower than it binds to apolypeptide of SEQ ID NO: 1. In one example, a protein of the presentdisclosure binds to a polypeptide of SEQ ID NO: 1 in which an alanine issubstituted for the leucine at a position 111 of SEQ ID NO: 1 at a levelat least about 60 fold lower than it binds to a polypeptide of SEQ IDNO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thehistidine at position 168 of SEQ ID NO: 1 at a level no more than 5 foldor 4 fold or 3 fold or 2 fold or 1 fold lower than it binds to apolypeptide of SEQ ID NO: 1.

In one example, a protein of the present disclosure binds to apolypeptide of SEQ ID NO: 1 in which an alanine is substituted for thelysine at position 167 of SEQ ID NO: 1 at a level no more than 5 fold or4 fold or 3 fold or 2 fold or 1 fold lower than it binds to apolypeptide of SEQ ID NO: 1.

The level of binding is conveniently determined using a biosensor.

The present disclosure contemplates any combination of the foregoingcharacteristics. In one example, a protein described herein has all ofthe binding characteristics set forth in the preceding seven paragraphs.

Epitope Mapping

In another example, the epitope bound by a protein described herein ismapped. Epitope mapping methods will be apparent to the skilled artisan.For example, a series of overlapping peptides spanning the hG-CSFRsequence or a region thereof comprising an epitope of interest, e.g.,peptides comprising 10-15 amino acids are produced. The protein is thencontacted to each peptide and the peptide(s) to which it bindsdetermined. This permits determination of peptide(s) comprising theepitope to which the protein binds. If multiple non-contiguous peptidesare bound by the protein, the protein may bind a conformational epitope.

Alternatively, or in addition, amino acid residues within hG-CSFR aremutated, e.g., by alanine scanning mutagenesis, and mutations thatreduce or prevent protein binding are determined. Any mutation thatreduces or prevents binding of the protein is likely to be within theepitope bound by the protein.

A further method is exemplified herein, and involves binding hG-CSFR ora region thereof to an immobilized protein of the present disclosure anddigesting the resulting complex with proteases. Peptide that remainsbound to the immobilized protein are then isolated and analyzed, e.g.,using mass spectrometry, to determine their sequence.

A further method involves converting hydrogens in hG-CSFR or a regionthereof to deutrons and binding the resulting protein to an immobilizedprotein of the present disclosure. The deutrons are then converted backto hydrogen, the hG-CSFR or region thereof isolated, digested withenzymes and analyzed, e.g., using mass spectrometry to identify thoseregions comprising deutrons, which would have been protected fromconversion to hydrogen by the binding of a protein described herein.

Optionally, the dissociation constant (Kd) of a protein for hG-CSFR oran epitope thereof is determined. The “Kd” or “Kd value” for a hG-CSFRbinding protein is in one example measured by a radiolabeled orfluorescently-labeled hG-CSFR binding assay. This assay equilibrates theprotein with a minimal concentration of labeled G-CSFR in the presenceof a titration series of unlabeled hG-CSFR. Following washing to removeunbound hG-CSFR, the amount of label is determined, which is indicativeof the Kd of the protein.

According to another example the Kd or Kd value is measured by usingsurface plasmon resonance assays, e.g., using BIAcore surface plasmonresonance (BIAcore, Inc., Piscataway, N.J.) with immobilized hG-CSFR ora region thereof.

In some examples, proteins having a similar Kd or a higher Kd than C1.2or C1.2G are selected, because they are likely to compete for binding tohG-CSFR.

Determining Competitive Binding

Assays for determining a protein that competitively inhibits binding ofmonoclonal antibody C1.2 or C1.2G will be apparent to the skilledartisan. For example, C1.2 or C1.2G is conjugated to a detectable label,e.g., a fluorescent label or a radioactive label. The labeled antibodyand the test protein are then mixed and contacted with hG-CSFR or aregion thereof (e.g., a polypeptide comprising SEQ ID NO: 1) or a cellexpressing same. The level of labeled C1.2 or C1.2G is then determinedand compared to the level determined when the labeled antibody iscontacted with the hG-CSFR, region or cells in the absence of theprotein. If the level of labeled C1.2 or C1.2G is reduced in thepresence of the test protein compared to the absence of the protein, theprotein is considered to competitively inhibit binding of C1.2 or C1.2Gto hG-CSFR.

Optionally, the test protein is conjugated to different label to C1.2 orC1.2G. This alternate labeling permits detection of the level of bindingof the test protein to hG-CSFR or the region thereof or the cell.

In another example, the protein is permitted to bind to hG-CSFR or aregion thereof (e.g., a polypeptide comprising SEQ ID NO: 1) or a cellexpressing same prior to contacting the hG-CSFR, region or cell withC1.2 or C1.2G. A reduction in the amount of bound C1.2 or C1.2G in thepresence of the protein compared to in the absence of the proteinindicates that the protein competitively inhibits C1.2 or C1.2G bindingto hG-CSFR. A reciprocal assay can also be performed using labeledprotein and first allowing C1.2 or C1.2G to bind to G-CSFR. In thiscase, a reduced amount of labeled protein bound to hG-CSFR in thepresence of C1.2 or C1.2G compared to in the absence of C1.2 or C1.2Gindicates that the protein competitively inhibits binding of C1.2 orC1.2G to hG-CSFR.

Any of the foregoing assays can be performed with a mutant form ofhG-CSFR and/or SEQ ID NO: 1 and/or a ligand binding region of hG-CSFR towhich C1.2 or C1.2G binds, e.g., as described herein.

Determining Neutralization

In some examples of the present disclosure, a protein is capable ofneutralizing hG-CSFR signaling.

Various assays are known in the art for assessing the ability of aprotein to neutralize signaling of a ligand through a receptor.

In one example, the protein reduces or prevents G-CSF binding to thehG-CSFR. These assays can be performed as a competitive binding assay asdescribed herein using labeled G-CSF and/or labeled protein.

In another example, the protein reduces formation of CFU-G when CD34⁺bone marrow cells are cultured in the presence of G-CSF. In such assays,CD34⁺ bone marrow cells are cultured in a semi-solid cell culture mediumin the presence of G-CSF (e.g., about 10 ng/ml cell culture medium) and,optionally stem cell factor (e.g., about 10 ng/ml cell culture medium)in the presence or absence of a test protein. After a sufficient timefor granulocyte clones (CFU-G) to form, the number of clones or coloniesis determined. A reduction in the number of colonies in the presence ofthe protein compared to in the absence of the protein indicates that theprotein neutralizes G-CSF signaling. By testing multiple concentrationsof the protein an IC₅₀ is determined, i.e., a concentration at which 50%of the maximum inhibition of CFU-G formation occurs. In one example, theIC₅₀ is 0.2 nM or less, such as 0.1 nM or less, for example, 0.09 nM orless, or 0.08 nM or less, or 0.07 nM or less, or 0.06 nM or less or 0.05nM or less. In one example, the IC₅₀ is 0.04 nM or less. In anotherexample, the IC₅₀ is 0.02 nM or less. The foregoing IC₅₀s relate to anyCFU-G assay described herein.

In a further example, the protein reduces proliferation of cells (e.g.,BaF3 cells) expressing hG-CSFR which are cultured in the presence ofG-CSF. Cells are cultured in the presence of G-CSF (e.g., 0.5 ng/ml) andthe presence or absence of a test protein. Methods for assessing cellproliferation are known in the art and include, for example, MTTreduction and thymidine incorporation. A protein that reduces the levelof proliferation compared to the level observed in the absence of theprotein is considered to neutralize G-CSF signaling. By testing multipleconcentrations of the protein an IC₅₀ is determined, i.e., aconcentration at which 50% of the maximum inhibition of cellproliferation occurs. In one example, the IC₅₀ is 6 nM or less, such as5.9 nM or less. In another example, the IC₅₀ is 2 nM or less or 1 nM orless or 0.7 nM or cell or 0.6 nM or less or 0.5 nM or less. Theforegoing IC₅₀s relate to any cell proliferation assay described herein.

In a further example, the protein reduces mobilization of hematopoieticstem cells and/or endothelial progenitor cells in vivo following G-CSFadministration and/or reduces the number of neutrophils in vivo, e.g.,following G-CSF administration (however this is not essential). Forexample, the protein is administered to a subject, optionally before, atthe time of or after administration of G-CSF or a modified form thereof(e.g., PEGylated G-CSF or filgrastim). The number of hematopoietic stemcells (e.g., expressing CD34 and/or Thy1) and/or endothelial progenitorcells (e.g., expressing CD34 and VEGFR2) and/or neutrophils (identifiedmorphologically and/or expressing e.g., CD10, CD14, CD31 and/or CD88) isassessed. A protein that reduces the level of the cell(s) compared tothe level observed in the absence of the protein is considered toneutralize G-CSF signaling. In one example, the protein reduces thenumber of neutrophils without inducing neutropenia.

Other methods for assessing neutralization of G-CSF signaling arecontemplated by the present disclosure.

Determining Effector Function

As discussed herein, some proteins of the present disclosure havereduced effector function. Methods for assessing ADCC activity are knownin the art.

In one example, the level of ADCC activity is assessed using a ⁵¹Crrelease assay, an europium release assay or a ³⁵S release assay. In eachof these assays, cells expressing G-CSFR are cultured with one or moreof the recited compounds for a time and under conditions sufficient forthe compound to be taken up by the cell. In the case of a ³⁵S releaseassay, cells expressing hG-CSFR can be cultured with ³⁵S-labeledmethionine and/or cysteine for a time sufficient for the labeled aminoacids to be incorporated into newly synthesized proteins. Cells are thencultured in the presence or absence of the protein and in the presenceof immune effector cells, e.g., peripheral blood mononuclear cells(PBMC) and/or NK cells. The amount of ⁵¹Cr, europium and/or ³⁵S in cellculture medium is then detected, and little or no change in the presenceof the protein compared to in the absence of protein (or a reduced levelof the compound compared to the level observed in the presence of ananti-hG-CSFR antibody comprising a human IgG1 Fc) indicates that theprotein has reduced effector function. Exemplary publications disclosingassays for assessing the level of ADCC induced by a protein includeHellstrom, et al. Proc. Natl. Acad. Sci. USA 83:7059-7063, 1986 andBruggemann, et al., J. Exp. Med. 166:1351-1361, 1987.

Other assays for assessing the level of ADCC induced by a proteininclude ACTI™ nonradioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. CA, USA) or CytoTox 96® non-radioactivecytotoxicity assay (Promega, Wis., USA).

C1q binding assays may also be carried out to confirm that the proteinis able to bind C1q and may induce CDC. To assess complement activation,a CDC assay may be performed (see, for example, Gazzano-Santoro et al,J. Immunol. Methods 202: 163, 1996.

Determining Half Life

Some proteins encompassed by the present disclosure have an improvedhalf-life, e.g., are modified to extend their half-life compared toproteins that are unmodified. Methods for determining a protein with animproved half-life will be apparent to the skilled person. For example,the ability of a protein to bind to a neonatal Fc receptor (FcRn) isassessed. In this regard, increased binding affinity for FcRn increasedthe serum half-life of the molecule (see for example, Kim et al., Eur JImmunol., 24:2429, 1994).

The half-life of a protein of the disclosure can also be measured bypharmacokinetic studies, e.g., according to the method described by Kimet al, Eur J of Immunol 24:542, 1994. According to this methodradiolabeled protein is injected intravenously into mice and its plasmaconcentration is periodically measured as a function of time, forexample at 3 minutes to 72 hours after the injection. The clearancecurve thus obtained should be biphasic, that is, an alpha phase and betaphase. For the determination of the in vivo half-life of the protein,the clearance rate in beta-phase is calculated and compared with that ofthe wild type or unmodified protein.

Assessing Therapeutic Efficacy

Assays for assessing therapeutic efficacy are described hereinabove inrelation to determining neutralization by a protein.

In another example, the efficacy of a protein to treat a condition isassessed using an in vivo assay.

For example, the protein is tested in an animal model of arthritis.Exemplary models include a SKG strain of mouse (Sakaguchi et al.,Nature, 426: 454-460), rat type II collagen arthritis model, mouse typeII collagen arthritis model or antigen induced arthritis models inseveral species (Bendele J Musculoskel Neuron Interact; 1(4):377-385,2001). In these assays, arthritis is induced and the ability of theprotein to reduce one or more symptoms of arthritis, e.g., jointinflammation and/or markers of inflammation in synovial fluid isassessed. A protein that reduces a symptom of arthritis is considereduseful for treating this condition or a G-CSF-mediated condition (e.g.,a G-CSF-mediated inflammatory condition).

The protein can also or alternatively be tested in a model of COPD,e.g., in which a non-human mammal (e.g., a rodent, such as, a mouse) isexposed to cigarette smoke. Following exposure, the mammal isadministered a protein and the level of lung inflammation and/or thenumber of neutrophils in the lung is assessed or estimated usingstandard techniques. A protein that reduces lung inflammation and/or thenumber of neutrophils is considered useful for treating lunginflammation or COPD or a G-CSF-mediated condition (e.g., aG-CSF-mediated inflammatory condition, such as a G-CSF-mediatedinflammatory lung condition).

Proteins described herein can also tested in in vivo models ofinflammatory neurological disease. Exemplary models include EAE modelsin which a mouse or rat is immunized with a myelin sheath protein orpeptide derived therefrom (e.g., MOG, MBP or PLP) and an immune responseis generated against the protein thereby inducing a model of MS.Alternatively, T cells that are immunoreactive with a myelin sheathprotein are introduced into mice or rats to induce EAE. Exemplary EAEmodels are reviewed in, for example Tsunoda and Fujinami, J NeuropatholExp Neurol. 55:673-686, 1996.

Other models of MS include transgenic animals expressing T cellreceptors specific for a myelin protein, e.g., MOG, MBP or PLP.Exemplary models are described, for example, in Bettelli et al., JEM197:1073-1081, 2003; Illés et al., Proc. Natl. Acad. Sci. USA, 101:11749-11754, 2004; or Rossi et al., J. Biomolecular Screening, 12:481-489, 2007; or are commercially available, e.g., from JacksonLaboratories USA (e.g. mice 2D2 having transgenic T cell receptorsreactive with MOG).

In a further example, a protein described herein according to anyexample is tested in a model of uveitis. Models of uveitis include thoseinduced by immunizing a non-human mammal with a protein such as retinalarrestin, recoverin or rhodopsin or administration of bacterialendotoxin to eye. Exemplary models of uveitis are described, forexample, in Caspi, Drug Discovery Today, 3: 3-9, 2006.

A protein of the disclosure can also be tested in models ofangiogenesis, e.g., Iris Pharma Inc's models of ocular angiogenesis, oran alginate encapsulated tumor cell model, and/or by assessing theability of a cancer cell to metastasize in a subject.

Conditions to be Treated

The present disclosure contemplates treatment or prevention of anycondition that is caused by or exacerbated by G-CSF in a subject. In oneexample, the condition is an autoimmune or inflammatory condition.

In one example, the inflammatory or autoimmune condition is aninflammatory joint condition, such as, inflammatory arthritis,rheumatoid arthritis or idiopathic arthritis, e.g., juvenile idiopathicarthritis. In one example, the condition is rheumatoid arthritis.

In one example, the inflammatory or autoimmune condition is aninflammatory eye condition. For example, the condition is uveitis.

In one example, the inflammatory or autoimmune condition is aninflammatory lung condition, such as, a pulmonary disease associatedwith neutrophil infiltration, e.g., COPD. In one example, the conditionis COPD.

In one example, the inflammatory or autoimmune condition is aninflammatory neurological condition, such as, Devic's disease, a viralinfection in the brain or multiple sclerosis. In one example, thecondition is multiple sclerosis, which includes chronic progressivemultiple sclerosis or relapsing-remitting multiple sclerosis.

In another example, the condition is cancer (including angiogenesisthereof) or metastasis thereof.

In one example, the subject is resistant to, does not adequately respondto, or is unsuitable for treatment with another compound used to treatthe condition. For example, the subject suffering from an autoimmune orinflammatory condition is resistant to, does not adequately respond to,or is unsuitable for treatment with a corticosteroid and/or animmunosuppressant and/or cyclophosphamide and/or methotrexate and/or ananti-TNF antibody or soluble TNF receptor and/or an anti-CD20 antibodyand/or an anti-IL6 antibody and/or an anti-CD22 antibody.

Compositions

In some examples, a protein as described herein can be administeredorally, parenterally, by inhalation spray, adsorption, absorption,topically, rectally, nasally, bucally, vaginally, intraventricularly,via an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, or by anyother convenient dosage form. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal, intraventricular, intrasternal, and intracranial injectionor infusion techniques.

Methods for preparing a protein into a suitable form for administrationto a subject (e.g. a pharmaceutical composition) are known in the artand include, for example, methods as described in Remington'sPharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa.,1990) and U.S. Pharmacopeia: National Formulary (Mack PublishingCompany, Easton, Pa., 1984).

The pharmaceutical compositions of this disclosure are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ or joint. Thecompositions for administration will commonly comprise a solution ofprotein dissolved in a pharmaceutically acceptable carrier, for examplean aqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of proteins of the presentdisclosure in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs. Exemplary carriers include water, saline, Ringer'ssolution, dextrose solution, and 5% human serum albumin. Nonaqueousvehicles such as mixed oils and ethyl oleate may also be used. Liposomesmay also be used as carriers. The vehicles may contain minor amounts ofadditives that enhance isotonicity and chemical stability, e.g., buffersand preservatives.

Upon formulation, proteins of the present disclosure will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically/prophylactically effective.Formulations are easily administered in a variety of dosage forms, suchas the type of injectable solutions described above, but otherpharmaceutically acceptable forms are also contemplated, e.g., tablets,pills, capsules or other solids for oral administration, suppositories,pessaries, nasal solutions or sprays, aerosols, inhalants, liposomalforms and the like. Pharmaceutical “slow release” capsules orcompositions may also be used. Slow release formulations are generallydesigned to give a constant drug level over an extended period and maybe used to deliver compounds of the present disclosure.

WO2002/080967 describes compositions and methods for administeringaerosolized compositions comprising antibodies for the treatment of,e.g., asthma, which are also suitable for administration of a protein ofthe present disclosure.

Combination Therapies

In one example, a protein of the present disclosure is administered incombination with another compound useful for treating a disease orcondition described herein, either as combined or additional treatmentsteps or as additional components of a therapeutic formulation.

For example, the other compound is an anti-inflammatory compound.Alternatively, or additionally, the other compound is animmunosuppressant. Alternatively, or additionally, the other compound isa corticosteroid, such as prednisone and/or prednisolone. Alternatively,or additionally, the other compound is methotrexate. Alternatively, oradditionally, the other compound is cyclophosphamide. Alternatively, oradditionally, the other compound is mycophenolate mofetil.Alternatively, or additionally, the other compound is an anti-CD20antibody (e.g., rituximab or ofatumumab). Alternatively, oradditionally, the other compound is an anti-CD22 antibody (e.g.,epratuzumab). Alternatively, or additionally, the other compound is ananti-TNF antibody (e.g., infliximab or adalimumab or golimumab) orsoluble TNF receptor (e.g., etanercept). Alternatively, or additionally,the other compound is a CTLA-4 antagonist (e.g., abatacept, CTLA4-Ig).Alternatively, or additionally, the other compound is an anti-IL-6antibody. Alternatively, or additionally, the other compound is a BLysantagonist, such as an anti-BLys antibody (e.g., belimumab).

In another example, the other compound is a chemotherapy drug or otherdrug used for treating cancer.

In another example, the protein described herein is administered beforeor after radiotherapy for the treatment of cancer.

Dosages and Timing of Administration

Suitable dosages of proteins of the present disclosure will varydepending on the specific protein, the condition to be treated and/orthe subject being treated. It is within the ability of a skilledphysician to determine a suitable dosage, e.g., by commencing with asub-optimal dosage and incrementally modifying the dosage to determinean optimal or useful dosage. Alternatively, to determine an appropriatedosage for treatment/prophylaxis, data from the cell culture assays oranimal studies are used, wherein a suitable dose is within a range ofcirculating concentrations that include the ED₅₀ of the active compoundwith little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. A therapeutically/prophylactically effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the compound whichachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma maybe measured, for example, byhigh performance liquid chromatography.

In some examples, a method of the present disclosure comprisesadministering a prophylactically or therapeutically effective amount ofa protein described herein.

The term “therapeutically effective amount” is the quantity which, whenadministered to a subject in need of treatment, improves the prognosisand/or state of the subject and/or that reduces or inhibits one or moresymptoms of a clinical condition described herein to a level that isbelow that observed and accepted as clinically diagnostic or clinicallycharacteristic of that condition. The amount to be administered to asubject will depend on the particular characteristics of the conditionto be treated, the type and stage of condition being treated, the modeof administration, and the characteristics of the subject, such asgeneral health, other diseases, age, sex, genotype, and body weight. Aperson skilled in the art will be able to determine appropriate dosagesdepending on these and other factors. Accordingly, this term is not tobe construed to limit the present disclosure to a specific quantity,e.g., weight or amount of protein(s), rather the present disclosureencompasses any amount of the protein(s) sufficient to achieve thestated result in a subject. In one example, a therapeutically effectiveamount of the protein does not induce neutropenia.

As used herein, the term “prophylactically effective amount” shall betaken to mean a sufficient quantity of a protein to prevent or inhibitor delay the onset of one or more detectable symptoms of a clinicalcondition. The skilled artisan will be aware that such an amount willvary depending on, for example, the specific protein(s) administeredand/or the particular subject and/or the type or severity or level ofcondition and/or predisposition (genetic or otherwise) to the condition.Accordingly, this term is not to be construed to limit the presentdisclosure to a specific quantity, e.g., weight or amount of protein(s),rather the present disclosure encompasses any amount of the protein(s)sufficient to achieve the stated result in a subject. In one example, aprophylactically effective amount of the protein does not induceneutropenia.

For in vivo administration of the proteins described herein, normaldosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of anindividual's body weight or more per day. Exemplary dosages and rangesthereof are described herein. For repeated administrations over severaldays or longer, depending on the severity of the disease or disorder tobe treated, the treatment can be sustained until a desired suppressionof symptoms is achieved.

In some examples, the protein is administered at an initial (or loading)dose of between about 1 mg/kg to about 30 mg/kg, such as from about 1mg/kg to about 10 mg/kg, or about 1 mg/kg or about 2 mg/kg or 5 mg/kg.The protein can then be administered at a lower maintenance dose ofbetween about 0.01 mg/kg to about 2 mg/kg, such as from about 0.05 mg/kgto about 1 mg/kg, for example, from about 0.1 mg/kg to about 1 mg/kg,such as about 0.1 mg/kg or 0.5 mg/kg or 1 mg/kg. The maintenance dosesmay be administered every 7-30 days, such as, every 10-15 days, forexample, every 10 or 11 or 12 or 13 or 14 or 15 days.

In some examples, the protein is administered at a dose of between about0.01 mg/kg to about 50 mg/kg, such as between about 0.05 mg/kg to about30 mg/kg, for example, between about 0.1 mg/kg to about 20 mg/kg, forexample, between about 0.1 mg/kg to about 10 mg/kg, such as betweenabout 0.1 mg/kg to about 2 mg/kg. For example, the protein isadministered at a dose of between about 0.01 mg/kg to about 5 mg/kg,such as from about 0.1 mg/kg to about 2 mg/kg, such as about 0.2 mg/kgor 0.3 mg/kg or 0.5 mg/kg or 1 mg/kg or 1.5 mg/kg (e.g., without ahigher loading dose or a lower maintenance dose). In some examples,numerous doses are administered, e.g., every 7-30 days, such as, every10-22 days, for example, every 10-15 days, for example, every 10 or 11or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 days.For example, the protein is administered every 7 days or every 14 daysor every 21 days.

In some examples, at the time of commencing therapy, the mammal isadministered the protein on no more than 7 consecutive days or 6consecutive days or 5 consecutive days or 4 consecutive days.

In the case of a mammal that is not adequately responding to treatment,multiple doses in a week may be administered. Alternatively, or inaddition, increasing doses may be administered.

In another example, for mammals experiencing an adverse reaction, theinitial (or loading) dose may be split over numerous days in one week orover numerous consecutive days.

Administration of a protein according to the methods of the presentdisclosure can be continuous or intermittent, depending, for example, onthe recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of a protein may beessentially continuous over a preselected period of time or may be in aseries of spaced doses, e.g., either during or after development of acondition.

Non-Limiting Examples Methods

The Ig and CRH domains of the G-CSFR are involved in ligand binding andreceptor dimerization (Layton et al., J. Biol Chem, 272: 29735-29741,1997 and Fukunaga et al, EMBO J. 10: 2855-2865 1991). Soluble forms ofG-CSFR (with either a C-terminal polyhistidine tag or an Fc sequence)comprising these portions of the receptor have been used in variousstudies of the receptor, and mutation of the free cysteines at positions78, 163, and 228 of the receptor assists in expression and isolation ofthe soluble receptor polypeptide (Mine et al., Biochem., 43: 2458-2464,2004) without affecting ligand binding. In the present studies solubleforms of the receptor comprising amino acids 25-335 of hG-CSFR withmutations C78A, C163S and C228S were generally used (e.g. SEQ ID NO:1)and the corresponding segment of cynoG-CSFR with the cysteine mutationswas generally used for studies on the cynomolgus monkey receptor.Various point mutations of the soluble receptor of SEQ ID NO:1 have alsobeen utilized. Reference to hG-CSFR-Fc means the polypeptide of SEQ IDNO:1 wherein the C-terminal polyhistidine tag has been replaced with anFc sequence. cynoG-CSFR-Fc means the corresponding segment of cynoG-CSFRwith an Fc sequence attached to its C-terminal In some instance thecorresponding extracellular domains of the wild type receptor have beenused, and in these instances it is specifically noted. The inventorshave shown that antibodies and proteins comprising antigen binding sitesthereof (e.g., Fab) bind to wild type hG-CSF polypeptides and to thesemutant proteins with highly similar affinity. Accordingly, studies usingthe mutant proteins are a model of studies using wild type hG-CSFR.

Identification of Fabs from Phage Display Library

A phage display library was screened for clones binding to hG-CSFR whicheluted upon addition of G-CSF ligand. Fabs were assessed for theirability to bind hG-CSFR, compete G-CSF binding, and cynomolgus G-CSFRcross-reactivity and some clones reformatted as IgG4 antibodies. Potencywas then tested through neutralization of G-CSF mediated proliferationin a BaF3 cell line stably transfected with hG-CSFR (described below)and to inhibit CFU-G formation in the presence of G-CSF.

Mammalian Expression Vector Construction for IgG Expression

Mammalian expression vectors were constructed using standard molecularbiology techniques by cloning the entire light chain (variable andconstant domains) and the variable region of the heavy chain from theselected phage-derived Fab constructs.

Cell Culture and Transient Transfection

Serum-free suspension adapted 293-T cells were obtained from GenechoiceInc. Cells were cultured in FreeStyle™ Expression Medium (Invitrogen)supplemented with penicillin/streptomycin/fungizone reagent(Invitrogen). Prior to transfection the cells were maintained at 37° C.in humidified incubators with an atmosphere of 8% CO₂.

The Transient transfection of the mammalian expression vectors using293-T cells was performed using 293fectin transfection reagent(Invitrogen) according to the manufacturer's instructions. The cellculture supernatants were harvested after 5 days incubation bycentrifugation at 2500 rpm and were then passed through a 0.45 μM filter(Nalgene) prior to purification using standard methods for IgGpurification.

Control Antibodies

Murine monoclonal antibodies 711, 744 and 774 (Layton et al., GrowthFactors, 14: 117-130, 1997) were used as control mouse antibodies.

Affinity Measurements of Fabs

To measure affinity of Fab for G-CSFR or G-CSFR-Fc, Fabs were expressedin E. coli and affinity measured using a Biacore 2000.

Measurement of Binding Kinetics for mAbs

Anti-human (Goat anti-human IgG (gamma) mouse adsorbed, Invitrogen, CatNo. H10500) or anti mouse Fc specific antibody (Jackson Immuno ResearchLabs inc. Cat No. 515-005-071) was chemically immobilized on a CM-5sensor surface using amine coupling chemistry.

The immobilized antibodies were then used to capture anti hG-CSFR mAbsfrom solution. Soluble hG-CSFR proteins (as described in the methodssection) were then injected over captured mAb at various concentrations.mAbs were captured for 180 seconds at 0.3 μg/ml. Soluble hG-CSFR at 0,1.25, 2.5, 5, 10, 20 and 40 nM (in duplicate) was injected for 10minutes and dissociation was monitored for 30 minutes. Responses from areference flow cell (in which mAb was not captured, but otherwisetreated identically), were subtracted. The responses from a blankinjection were then subtracted from the resultant sensorgrams.

The final corrected responses were fitted using non-linear regression toa model describing 1:1 kinetics, including a term for mass transportlimitation. The Rmax value was fitted locally, to account for slightdeviations in the level of mAb captured. Equilibrium dissociationconstant (KD) was determined

Kinetic Analysis of Fab C1.2, 5D11, 711 and 744

Fab fragments were generated by papain digestion where 3 mg of antibodywas digested (1:500) with pre-activated papain for 40 minutes as perinstructions using a papain digestion kit (Sigma, USA). Resultant Fabwas purified, by adsorption, away from residual Fc and undigestedantibody using protein A purification (mAbSelect, GE, Sweden).

Duplicate biosensor analysis of the Fab were performed using a Biacore2000 (GE, Sweden) with a doubling dilution of Fab antibodies (100 nM to0.39 nM in 0.1 mg/ml BSA) at a flow rate of 30 μl/min. Binding (100 μl)was monitored to respective flow cells containing control immobilisedblank, immobilized cyno G-CSFR-Fc, immobilized human G-CSFR-Fc andimmobilized human G-CSFR. Receptor proteins (20 μg per ml in 20 mMSodium Acetate pH 4.5) were previously immobilized to a CMS chip using aNHS/EDC coupling kit as per the manufacturer's instructions (Biacore GE,Sweden). Target immobilization values were set at 700, 700 and 500resonance units for cyno G-CSFR-Fc, hG-CSFR-Fc and hG-CSFR,respectively. Chip immobilisation was quenched with 50 mM ethanolaminepH 8.0. Dissociation of surface binding was monitored for 1000 secondsprior to desorption of the remaining complex using 50 mM Phosphoricacid. Reference binding was then subtracted from the control channel andkinetics generated using biaevaluation software on the Biacore 2000.

Duplicate biosensor analysis of the antibodies c1.2 and 5D11 in IgG4format were performed using a Biacore 2000 with a doubling dilution ofthe antibodies (312 nM to 2.4 nM in 0.1 mg/ml BSA) at a flow rate of 30ul/min. Binding (100 μl) was monitored to respective flow cellscontaining control immobilized blank, immobilized cyno-G-CSFR-Fc,immobilized hG-CSFR-Fc and immobilized hG-CSFR. Receptor proteins (20 ugper ml in 20 mM Sodium Acetate pH 4.5) were previously immobilized to aCMS chip using a NHS/EDC coupling kit as per the manufacturer'sinstructions (Biacore GE, Sweden). Target immobilization values were setat 700, 700 and 500 resonance units for cyno-G-CSFR-Fc, hG-CSFR-Fc andhG-CSFR, respectively. Chip immobilization was quenched with 50 mMethanolamine pH 8.0. Dissociation of surface binding was monitored for1000 seconds prior to desorption of the remaining complex using 50 mMPhosphoric acid. Reference binding was then subtracted from the controlchannel and kinetics generated using biaevaluation software on theBiacore 2000.

BIAcore mAb Kinetics of Affinity Matured C1.2G Antibodies

Anti human (Goat anti Human IgG (gamma) mouse adsorbed, Invitrogen, CatNo. H10500) was chemically immobilised on a CM-5 sensor surface usingamine coupling chemistry and then used to capture the C1.2G affinitymatured anti hG-CSFR mAbs at 1 mg/ml for 3 mins. Soluble hG-CSFR wasthen injected over the captured mAb at 0, 10 and 40 nM. Soluble hG-CSFRwas injected for 5 minutes and dissociation was monitored for 30minutes. Responses from a reference flow cell (in which mAb was notcaptured, but otherwise treated identically), were subtracted. Theresponses from a blank injection were then subtracted from the resultantsensorgrams.

The final corrected responses were fitted using non-linear regression toa model describing 1:1 kinetics, including a term for mass transportlimitation. The Rmax value was fitted locally, to account for slightdeviations in the level of mAb captured. Association rate (ka),dissociation rate (kd) and equilibrium dissociation constant (K_(D))were determined

hG-CSFR/BaF3 Proliferation Bioassay—MTT Reduction

BaF3 cells expressing hG-CSFR were obtained from the Ludwig InstituteMelbourne. To assess the inhibition of G-CSF mediated proliferation byanti hG-CSFR antibodies, serial dilutions of antibody were added to2×10⁴ cells/well in DME medium with 5% FCS and 0.5 ng/ml hGCSF in 96well plates and incubated for 48 hours at 37° C., 10% CO₂. Cellproliferation was determined by MTT reduction and measured by absorbanceat 490 nM.

hG-CSFR/BaF3 Proliferation Bioassay—3H-Thymidine Incorporation

BAF/3 cells engineered to express human G-CSFR and which proliferate inresponse to human G-CSF were used to measure the ability of variousmonoclonal antibodies to neutralize the activity of G-CSF. Cells wereplated at 1×10⁴ cells in 96 well plates in RPMI/105FCS in the presenceof 10 ng/mL human G-CSF and increasing concentrations of variousanti-G-CSFR monoclonal antibodies for 48 hours at 37° C. Cells werepulsed with ³H-thymidine for the last 6 hours of culture before beingharvested onto glass fibre filters and the level of radioactivethymidine incorporated into DNA determined by liquid scintillationcounting.

Human CFU-G Progenitor Bioassay

CD34⁺ bone marrow cells were incubated in semi-solid medium in thepresence of 10 ng/ml stem cell factor, 10 ng/ml hG-CSF and titratingconcentrations of test antibody. CFU-G were enumerated after 14 days ofculture.

Epitope Comparison—Competition Binding

This design of this experiment is built on the premise that for 2antibodies to be capable of simultaneously binding a single molecule,the epitopes of those 2 antibodies must be different.

Soluble G-CSFR of SEQ ID NO:1 was captured from solution by a surfaceimmobilized antibody. A second antibody was then injected over thecomplex. Responses from a reference flow cell (in which soluble hG-CSFRwas not captured, but otherwise treated identically), were subtracted.Binding of the second antibody indicates that the epitopes of the 2antibodies differ.

Responses measured at the end of the antibody binding phase were dividedby the response at the end of the hG-CSFR capture phase, to correctantibody binding level for the amount of hG-CSFR captured. These capturecorrected responses were then used to compare the binding of eachantibody to hG-CSFR in the presence of the other antibodies.

Antibodies C1.2, 5D11, 711 and 744 were chemically immobilized on a CM-5sensor surface using amine coupling chemistry. Soluble hG-CSFR wascaptured at 100 nM for 180 seconds. Each of the antibodies was theninjected in duplicate over captured hG-CSFR at 100 nM for 180 seconds. Ablank injection of buffer only was also performed.

Epitope Mapping of C1.2G, 711, 744 and 774

A series of alanine point mutations of SEQ ID NO:1 were generated,expressed in HEK293 cells and then purified. The binding affinity ofthese mutants for antibodies C1.2, 744 and 774 was measured, andcompared to that of SEQ ID NO:1. If a mutation resulted in a change ofaffinity by more than a factor of 2 from that of SEQ ID NO:1, thatresidue was deemed to contribute to the binding interaction, and thus islikely to be in or near the epitope. A third mAb (711) with an epitopeseparate and distinct to C1.2, 744 and 774, was included as a control inorder to account for any major structural changes brought about by themutations.

Anti human (Goat anti Human IgG (gamma) mouse adsorbed, Invitrogen, CatNo. H10500) or anti mouse Fc specific antibody (Jackson Immuno ResearchLabs inc. Cat No. 515-005-071) was chemically immobilized on a CM-5sensor surface using amine coupling chemistry. The immobilizedantibodies were then used to capture anti hG-CSFR mAbs from solution.Wild-type hG-CSFR ligand binding domain (SEQ ID NO:1) and each alaninepoint mutant were then injected over captured mAbs at variousconcentrations. Responses from a reference flow cell (in which mAb wasnot captured, but otherwise treated identically), were subtracted. Theresponses from a blank injection were then subtracted from the resultantsensorgrams.

The final corrected responses were fitted using non-linear regression toa model describing 1:1 kinetics, including a term for mass transportlimitation. The Rmax value was fitted locally, to account for slightdeviations in the level of mAb captured. Association rate (ka),dissociation rate (kd) and equilibrium dissociation constant (KD) weredetermined

C1.2 germline mAb was captured at 0.3 ug/ml for 180 sec, 711 at 1 μg/mlfor 180 sec, and 744 and 774 at 5 μg/ml for 180 sec.

For C1.2 and 744 kinetics, WT hG-CSFR and each ala mutant were injectedat 0, 2, 10, 50 and 250 nM for 300 sec and dissociation monitored for afurther 1800 sec.

For antibody 774 kinetics, WT hG-CSFR and each ala mutant were injectedat 0, 2, 10, 50 and 250 nM for 300 sec and dissociation monitored for afurther 600 sec.

For antibody 711 kinetics, WT hG-CSFR and each ala mutant were injectedat 0 and 100 nM for 180 sec and dissociation monitored for a further 180sec.

Example 1 Fully Human Anti-hG-CSFR Antibodies are Potent InhibitorsG-CSF Signaling

Using affinity measurements and the BaF3 proliferation assay describedabove, antibodies 711 and 744 were assessed for affinity to hG-CSFR andG-CSF neutralization assays. Antibody 711 was found to bind tohG-CSFR-Fc fusion (based on SEQ ID NO:1 as discussed in the methods)with an affinity greater than antibody 744 (K_(D) of 0.86 nM and 8.7 nM,respectively). Using the MTT-based bioassay described above, antibody711 was also found to more potently inhibit G-CSF-mediated cellproliferation than antibody 744 (IC₅₀ (nM G-CSF) of 8.8 nM and 2.4 nM,respectively).

Using the ³H-thymidine incorporation assay, antibody 711 was found toinhibit G-CSF-mediated cell proliferation with an IC₅₀ of 10.1 mg/ml;antibody 774 was found to inhibit G-CSF-mediated cell proliferation withan IC₅₀ of 37.4 μg/ml and the IC₅₀ for antibody 744 was not determinable(see FIG. 1).

Human antibodies isolated from a phage display library (antibodies C1.2and 5D11) and mouse monoclonal antibody 711 were assessed for theirability to inhibit G-CSF-mediated proliferation of BaF3 cells using thebioassay described above. Results showed that antibody C1.2 inhibitedBaF3 proliferation with an IC₅₀ of 0.5 nM; antibody 5D11 inhibitedproliferation an IC₅₀ of 5.9 nM; and 711 inhibited proliferation with anIC₅₀ of 3.4 nM.

The antibodies were also assessed for their ability to reduce or inhibitCFU-G formation by CD34⁺ bone marrow cells in the presence of G-CSF asdescribed above. Results showed that antibody C1.2 inhibited CFU-Gformation with an IC₅₀ of 0.016 nM; antibody 5D11 inhibited CFU-Gformation with an IC₅₀ of 0.039 nM; and 711 inhibited CFU-G formationwith an IC₅₀ of 0.411 nM.

Based on these assays, the human antibodies C1.2 and 5D11 are morepotent than 711 in inhibiting CFU-G formation and C1.2 is the mostpotent antibody in both bioassays.

Example 2 Affinity of Antibodies for Human G-CSFR and Cynomolgus MonkeyG-CSFR

Affinities of Fabs and IgG4 forms of 5D11 and C1.2 and Fabs of 711 and744 were also assessed to determine their affinities for the ligandbinding domain of hG-CSFR(SEQ ID NO:1), hG-CSFR-Fc and cynomolgus monkeyG-CSFR-Fc (based on SEQ ID NO:1 as discussed in the methods). In theseassays, the regions of G-CSFR were immobilized and binding of theindicated antibody or fragment to the immobilized polypeptide determinedas described in more detail in the general methods (Section entitled“Kinetic analysis of Fab C1.2, 5D11, 711 and 744”). Results are shown inTable 1.

TABLE 1 Affinities of 5D11 and C1.2 for human and cynomolgus monkeyG-CSFR Chip Immobilised with: Antibody/Fab hG-CSFR hG-CSFR-Fc cynoG-CSFR-Fc 5D11 Fab 1.30 nM 1.20 nM 0.42 nM C1.2 Fab 0.37 nM 0.33 nM 0.39nM 5D11 IgG4 65 pM 61 pM 37 pM C1.2 IgG4 27 pM 77 pM 54 pM

These data show that 5D11 and C1.2 have high affinities for hG-CSFR andthat these affinities are improved when the Fabs are expressed ascomplete IgG4 antibodies. Moreover, the affinities for hG-CSFR andcynoG-CSFR for 5D11 or C1.2 are similar. C1.2 has higher affinity forhG-CSFR than 5D11.

The Fab of antibody 711 was shown to have an affinity for hG-CSFR-Fc of0.86 nM and for cynoG-CSFR of 1.8 μM. The Fab of antibody 744 was shownto have an affinity for hG-CSFR-Fc of 8.7 nM and for cynoG-CSFR of >10μM. Thus, these Fabs have much poorer affinity for cynoG-CSFR than theydo for hG-CSFR.

Reciprocal assays were also performed (i.e., in which antibodies wereimmobilized and binding of wild type h-GCSFR or wild type cynoG-CSFR tothe immobilized antibody was determined in accordance with the generalmethods (Section entitled “Measurement of Binding Kinetics for mAbs”).Representative results are shown in Table 2.

TABLE 2 Representative affinities of antibodies C1.2G, 5D11, 711, 744and 774 for wild type hG-CSFR or wild type cynoG-CSFR. Binding to wtBinding to wt Captured Antibody hG-CSFR (K_(D)) cyno G-CSFR (K_(D))C1.2G (human IgG4) 1.4 nM 494 pM 5D11 (human IgG4) 6.09 nM 477 pM mAb711 (mIgG1) 1.69 nM 97 nM mAb744 (mIgG2a) 7.82 nM Negligible bindingmAb774 (mIgG1) 23.4 nM Negligible binding

These data shown that C1.2G and 5D11 bind to wild type cynoG-CSFR withhigher affinities than 711, 744 and 774. Moreover, the affinity of C1.2Gfor wild type hG-CSFR and wild type cynoG-CSFR are within about 3 foldof one another and the affinity of 5D11 for wild type hG-CSFR and wildtype cynoG-CSFR are within about 13 fold of one another.

Example 3 Germlining of C1.2

To minimize potential immunogenicity, the variable region framework ofC1.2 was changed to match that of the closest human germline framework.This required a single change in the framework of the heavy chain andfive changes in the light chain and resulted in a V_(H) with a sequenceset forth in SEQ ID NO: 4 and a V_(L) with a sequence set forth in SEQID NO: 5. Affinity of the germlined antibody (C1.2G) for G-CSFR wassimilar to C1.2 (k_(a) 9.54×10⁴±5.5×10³; k_(d) 1.31×10⁻⁴±2.6×10⁻⁶; K_(D)1.37±0.07 (N=8)). Affinity of C1.2G for hG-CSFR expressed on the cellsurface of BaF3 cells was shown to be 257 pM.

Using the ³H-thymidine incorporation assay described above, antibodyC1.2G was found to inhibit G-CSF-mediated cell proliferation with anIC₅₀ of 0.8 μg/ml (FIG. 1).

Reformatting of this antibody into a stabilized IgG4 produced anantibody comprising a heavy chain with a sequence set forth in SEQ IDNO: 64 and a light chain with a sequence set forth in SEQ ID NO: 65.

When expressed in CHO cells a lysine variant of the antibody wasobserved, i.e., in which one or both of the heavy chains lacked aC-terminal lysine residue (i.e., thus comprising a sequence set forth inSEQ ID NO: 64).

Example 4 Epitope Mapping Competition Binding

Published data has shown that mAb711 and mAb744 bound to differentdomains of the hG-CSFR. Competition binding experiments showed thatmAb711 but not mAb744 was able to bind to the hG-CSFR subsequent to thebinding of C1.2 Ab suggesting that C1.2 binds to a similar region of thereceptor as mAb744 but a different region to that of mAb711.

Epitope Excision to Identify Peptides Involved in C1.2 Binding

Epitope excision followed by mass spectrometry analysis was used toidentify four peptides of the hG-CSFR that were involved in the bindingof C1.2. In this method, the hG-CSFR protein is first bound to animmobilized C1.2 antibody and then digested with proteolytic enzymes.The bound peptides are then identified by MALDI and Electrospray massspectrometry. The four peptides identified by this approach mapped topositions 111-115, 170-176, 218-234 and/or 286-300 of hG-CSFR (SEQ IDNO: 1).

Binding of C1.2 and mAb744 to hG-CSFR Region Mutants

Published data (Tamada et al Proc Natl Acad Sci USA. 103:3135-3140,2006; Aritomi et al Acta Crystallogr D Biol Crystallogr; 56:751-7531999) has identified surface residues on the hG-CSFR. A number of theseresidues located within the four peptides identified by epitope excisionexperiments were substituted by alanine and the resulting mutant formsof a region of hG-CSFR were expressed and purified. The binding of C1.2and mAb744 to each of these mutants was assessed and the key residuesinvolved in binding identified. Results are shown in FIG. 2A. Alaninesubstitution of residues K167 and H168 resulted in a complete loss ofbinding by mAb744 whilst binding of C1.2 was unaffected. In contrast,alanine substitution of residue R287 resulted in a complete loss ofbinding by C1.2 with no effect on mAb744 binding. Other residues thatsignificantly reduced the binding of C1.2 were L111, L171, Y172, M198and H237. MAb744 bound these mutants at a similar affinity to that ofthe wild type receptor.

The above assay was repeated with the same antibodies together withmAb774/As shown in FIG. 2B, alanine substitution of residues K167, H168and L169 resulted in a complete loss of binding by mAb774 whilst bindingof C1.2 was unaffected. As with mAb 744, alanine substitution of residueL111, Y172, H237 and R287 had little or no effect on mAb774 binding.

Example 5 Affinity Maturation of C1.2G

Affinity maturation was performed by mutating residues in HCDR3 and/orLCDR3 of C1.2G and screening for Fabs that bound to hG-CSFR. Librariesof mutant antibodies were panned using biotinylated hG-CSFR-Fcrecombinant protein, either at a constant concentration over severalpanning rounds or at reducing concentrations.

At the completion of panning, a number of phage clones were selectedfrom each enriched library and sequenced. Unique clones were thenselected based on sequence and reformatted into fully human IgG4/kappaantibodies for binding analysis to hG-CSFR using Biacore (following thegeneral methods in the Section entitled “Measurement of Binding Kineticsfor mAbs”) and, in some cases ability to inhibit G-CSF-mediatedproliferation of BaF3 cells. The reformatted antibodies with improvedaffinities as compared to the parental C1.2G mAb are listed in Table 3.

TABLE 3 Characteristics of affinity matured antibodies LCDR3 HCDR3 IC50mAb Sequence Sequence³ KD (M)  (nM) C1.2G-987 IQYPQM¹ LGQSSA 4.46E−110.88 C1.2G-95 WEYPLV¹ wt 5.78E−11 0.26 C1.2G-79 QVSWEY² wt 6.13E−11 0.31C1.2G-83 WMYALF¹ wt 6.60E−11 0.21 C1.2G-1003 WHYPLT¹ LGSGST 6.82E−110.26 C1.2G-44 YSYPQK¹ wt 7.33E−11 0.39 C1.2G-97 FMYPLY¹ wt 9.06E−11 0.21C1.2G-986 YAYPQQ¹ LGFFQE 9.11E−11 0.28 C1.2G-56 YMYPIK¹ wt 9.93E−11 0.22C1.2G-77 EQGWNY² wt 1.07E−10 0.23 C1.2G-54 MWMPMG¹ LGMFLE 1.10E−10 0.50C1.2G-802 HFSMQY² wt 1.11E−10 0.26 C1.2G-967 WAYGLS¹ LGMYDL 1.34E−100.29 C1.2G-989 FYYPFY¹ LGQYMF 1.44E−10 0.34 C1.2G-63 ANSWGY² wt 1.61E−100.26 C1.2G-1002 WTYGQT¹ LGMYMN 1.67E−10 0.18 C1.2G-994 LEYPQM¹ LGQFMD1.70E−10 0.49 C1.2G-969 FQYAQH¹ LGQYQF 1.81E−10 0.41 C1.2G-1000 WMYAHM¹LGQMMY 1.82E−10 0.34 C1.2G-94 WVYPAW¹ wt 1.89E−10 ND C1.2G-975 WQIKLK¹LGQSML 2.09E−10 ND C1.2G-75 EESMNY² wt 2.11E−10 ND C1.2G-814 SQSMEY² wt2.21E−10 ND C1.2G-973 FKYPMT¹ LGQMVY 2.30E−10 ND C1.2G-977 WVYHLP¹LGEIRE 2.46E−10 ND C1.2G-984 IEYPAH¹ LGMMQS 2.50E−10 ND C1.2G-61 QQGMWM²wt 2.57E−10 ND C1.2G-852 wt LGHSLA 2.92E−10 ND C1.2G-996 MWMPIF¹ LGQYMG3.36E−10 ND C1.2G-43 IGYPGS¹ LGQFMR 3.36E−10 ND C1.2G-999 WEYAMF¹ LGMFHK3.56E−10 ND C1.2G-870 WMYHKI¹ wt 3.67E−10 ND C1.2G-877 FRYPFY¹ wt5.08E−10 ND C1.2G wt wt 6.33E−10 0.32 ¹Sequence is preceded by thesequence QQS ²Sequence is followed by the sequence PLT ³Sequence (otherthan wt) is preceded by the sequence LGE wt - sequence of CDR3 fromC1.2G ND - not determined

Example 6 C1.2G Reduces Neutrophil Levels without Inducing Neutropenia

Cynomolgus monkeys were administered pegylated G-CSF and C1.2G (10mg/kg) administered 12 hours later. As shown in FIG. 3, C1.2significantly reduced the level of neutrophils compared to controlanimals, however did not induce neutropenia.

Similar experiments dosing cynomolgus monkeys with between 0.1 to 10mg/kg of C1.2G 2 hours prior to administration with G-CSF also reducedthe level of neutrophils compared to control animals, however did notinduce neutropenia.

1. A protein comprising an antigen binding site of an antibody, whereinthe antigen binding site binds to human granulocyte-colony stimulatingfactor receptor (hG-CSFR) and neutralizes granulocyte-colony stimulatingfactor (G-CSF) signaling, and wherein the protein inhibits growth ofcolony forming units—granulocytes (CFU-G) from CD34⁺ bone marrow cellsgrown in the presence of G-CSF with an IC₅₀ of at least about 0.2 nM. 2.The protein of claim 1, wherein the antigen binding site binds to bothhuman and cynomolgus monkey granulocyte-colony stimulating factorreceptor (G-CSFR) with a similar affinity and neutralizesgranulocyte-colony stimulating factor (G-CSF) signaling.
 3. The proteinof claim 1 comprising at least a heavy chain variable region (V_(H)) anda light chain variable region (V_(L)), wherein the V_(H) and V_(L) bindto form a Fv comprising an antigen binding domain.
 4. The protein ofclaim 3, wherein the V_(H) and the V_(L) are in a single polypeptidechain or wherein the V_(L) and V_(H) are in separate polypeptide chains.5. The protein of claim 4, wherein if the V_(H) and V_(L) are in thesame polypeptide chain, the protein is: (i) a single chain Fv fragment(scFv); (ii) a dimeric scFv (di-scFv); or (iii) at least one of (i)and/or (ii) linked to a constant region of an antibody, a Fc or a heavychain constant domain (C_(H)) 2 and/or C_(H)3; or if the V_(H) and V_(L)are in separate polypeptide chains, the protein is: (i) a diabody; (ii)a triabody; (iii) a tetrabody; (iv) a Fab; (v) a F(ab′)₂; (vi) a Fv;(vii) an antibody; or (viii) one of (i) to (vi) linked to a constantregion of an antibody, a Fc or a heavy chain constant domain (C_(H)) 2and/or C_(H)3.
 6. The protein of claim 1 which is chimeric,de-immunized, humanized, human or primatized.
 7. The protein of claim 1,which inhibits G-CSF-induced proliferation of a BaF3 cell expressinghG-CSFR with an IC₅₀ of at least about 0.5 nM.
 8. The protein of claim1, which binds to a polypeptide comprising amino acids 1 to 311 of SEQID NO: 1 expressed as a fusion with an antibody Fc region with anaffinity of at least about 0.5 nM, wherein affinity is determined in anassay in which the polypeptide immobilized and the protein or antibodycontacted to the immobilized polypeptide.
 9. The protein of claim 1,which binds to hG-CSFR expressed on the surface of a cell at an affinityof at least about 1 nM.
 10. The protein of claim 1 conjugated to anothercompound.
 11. A composition comprising the protein of claim 1 and apharmaceutically acceptable carrier.
 12. A method for treating orpreventing a granulocyte-colony stimulating factor (G-CSF)-mediatedcondition in a subject, the method comprising administering the proteinof claim 1 to the subject.
 13. The method of claim 12 comprisingadministering an amount of the protein or antibody sufficient to reducethe number of neutrophils in a subject without inducing neutropenia. 14.The method of claim 13, wherein the amount of the protein is between0.05 mg/kg and 30 mg/kg.